Anti-Alpha-V Immunoliposome Composition, Methods, and Uses

ABSTRACT

An immunoliposome composition targeted to the alpha-V-integrin subunit of integrin receptors comprised of ligand-targeted liposomes bearing at least one targeting-ligand derived from an antibody and having binding specificity for at least one integrin receptor comprising an alpha-V subunit including αvβ1, αvβ3 αvβ5, αvβ6, or αvβ8 integrin cell receptors is described. The antibody-derived targeting ligand may be a Fab′ fragment, a scFv, or a the like. Binding of the immunoliposome to αv-integrin expressing cells, preferably results in internalization of the immunoliposome for cytoplasmic delivery of a liposome-entrapped agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/917,586, filed 11 May 2007, the contents of which are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The subject matter described herein relates to a liposome compositionhaving specific binding activity for alpha-V-integrin receptors. Thecomposition is intended for use in treating conditions characterized bycells that express any alpha-V-comprising integrin, such as αvβ3, αvβ5,and αvβ6 receptors.

BACKGROUND

Integrins are a superfamily of cell adhesion receptors, which exist asheterodimeric transmembrane glycoproteins. They are part of a largefamily of cell adhesion receptors which are involved incell-extracellular matrix and cell-cell interactions. Integrins playcritical roles in cell adhesion to the extracellular matrix (ECM) which,in turn, mediates cell survival, proliferation and migration throughintracellular signaling. The receptors consist of two subunits that arenon-covalently bound. Those subunits are called alpha (α) and beta (β).The alpha subunits all have some homology to each other, as do the betasubunits. The receptors always contain one alpha chain and one betachain and are thus called heterodimeric. Both of the subunits contributeto the binding of ligand. Eighteen alpha subunits and eight betasubunits have been identified, which heterodimerize to form at least 24distinct integrin receptors.

Among the variety of alpha chain subunits is a protein chain referred toas alpha V. The ITGAV gene encodes integrin alpha chain V (vitronectinreceptor, alpha-v; αv, antigen CD51). The I-domain containing integrinalpha-v undergoes post-translational cleavage to yield disulfide-linkedheavy and light chains, that combine with multiple integrin beta chainsto form different integrins. Alternative splicing of the gene yieldsseven different transcripts; a, b, c, e, f, h, j altogether encoding sixdifferent protein isoforms of alpha-V. Among the known associating betachains (beta chains 1, 3, 5, 6, and 8; ‘ITGB1’, ‘ITGB3’, ‘ITGB5’,‘ITGB6’, and ‘ITGB8’), each can interact with extracellular matrixligands. The alpha V beta 3 integrin, perhaps the most studied of these,is referred to as the vitronectin receptor (VNR). In addition toproviding for cell attachment to other cells or to extracellularproteins such as vitronectin (αvβ3) and fibronectin (αvβ6), theintegrins are capable of intracellular signaling which provides cluesfor cell migration and secretion of or elaboration of other proteinsinvolved in cell motility and invasion and angiogenesis. The alpha-vintegrin subfamily of integrins recognize the ligand motif arg-gly-asp(RGD) present in fibronection, vitronection, VonWillebrand factor, andfibrinogen. The alpha-V integrins are receptors for vitronectin,cytotactin, fibronectin, fibrinogen, laminin, matrixmetalloproteinase-2, osteopontin, osteomodulin, prothrombin,thrombospondin and von Willebrand factor. In case of HIV-1 infection,the interaction with extracellular viral Tat protein seems to enhanceangiogenesis in Kaposi's sarcoma lesions.

It has been established that integrins which are alpha-v containingheterodimers, particularly alpha-v/beta-6, the receptor for fibronectin,are involved in adhesion of carcinoma cells to fibronectin andvitronectin. This is especially true for carcinoma cells arising fromthe malignant progression of colon cancer (Lehmann, M. et al., CancerRes., 54(8):2102-7 (1994)). Furthermore, integrin expression in coloncancer cells is regulated by the cytoplasmic domain of the beta-6integrin subunit which signals through the ERK2 pathway (Niu, J. et al.,Int. J. Cancer, 99(4):529-537 (2002)) and beta6 expression is associatedwith secretion of gelatinase B, an enzyme involved in tumor cellinvasion and metastatic mechanisms (Agrez et al., Int. J. Cancer,81(11):90-97 (1999)).

There is now considerable evidence that progressive tumor growth isdependent upon angiogenesis, the formation of new blood vessels, toprovide tumors with nutrients and oxygen, to carry away waste productsand to act as conduits for the metastasis of tumor cells to distantsites (Gastl, G. et al., Oncol., 54(3):177-184 (1997)). Recent studieshave further defined the roles of integrins in the angiogenic process.During angiogenesis, a number of integrins that are expressed on thesurface of activated endothelial cells regulate critical adhesiveinteractions with a variety of ECM proteins to regulate distinctbiological events such as cell migration, proliferation anddifferentiation. Specifically, the closely related but distinctintegrins αvβ3 and αvβ5 have been shown to mediate independent pathwaysin the angiogenic process. An antibody generated against αvβ3 blockedbasic fibroblast growth factor (bFGF) induced angiogenesis, whereas anantibody specific to αvβ5 inhibited vascular endothelial growth factor(VEGF) induced angiogenesis (Eliceiri et al., J. Clin. Invest.,103:1227-1230 (1999); Friedlander et al., Science, 270:1500-1502(1995)). Therefore, integrins, and especially the alpha V subunitcontaining integrins, are a therapeutic targets for diseases thatinvolve angiogenesis, such as diseases of the eye and neoplasticdiseases, tissue remodeling such as restenosis, and proliferation ofcertain cells types, particularly epithelial and squamous cellcarcinomas.

Liposomes are spherical vesicles comprised of concentrically orderedlipid bilayers that encapsulate an aqueous phase. Liposomes serve as adelivery vehicle for therapeutic agents contained in the aqueous phaseor in the lipid bilayers. Delivery of drugs in liposome-entrapped formcan provide a variety of advantages, depending on the drug, including,for example, a decreased drug toxicity, altered pharmacokinetics, orimproved drug solubility. Liposomes when formulated to include a surfacecoating of hydrophilic polymer chains, so-called Stealth® orlong-circulating liposomes, offer the further advantage of a long bloodcirculation lifetime, due in part to reduced removal of the liposomes bythe mononuclear phagocyte system. Often an extended lifetime isnecessary in order for the liposomes to reach their desired targetregion or cell from the site of injection.

Targeted liposomes have targeting ligands or affinity moieties attachedto the surface of the liposomes. The targeting ligands may be antibodiesor fragments thereof, in which case the liposomes are referred to asimmunoliposomes. When administered systemically targeted liposomesdeliver the entrapped therapeutic agent to a target tissue, region or,cell. Because targeted liposomes are directed to a specific region orcell, healthy tissue is not exposed to the therapeutic agent. Suchtargeting ligands can be attached directly to the liposomes' surfaces bycovalent coupling of the targeting ligand to the polar head groupresidues of liposomal lipid components (see, for example, U.S. Pat. No.5,013,556). This approach, however, is suitable primarily for liposomesthat lack surface-bound polymer chains, as the polymer chains interferewith interaction between the targeting ligand and its intended target(Klibanov, A. L., et al., Biochim. Biophys. Acta., 1062:142-148 (1991);Hansen, C. B., et al., Biochim. Biophys. Acta, 1239:133-144 (1995)).

Alternatively, the targeting ligands can be attached to the free ends ofthe polymer chains forming the surface coat on the liposomes (Allen. T.M., et al., Biochim. Biophys. Acta, 1237:99-108 (1995); Blume, G. etal., Biochim. Biophys. Acta, 1149:180-184 (1993)). In this approach, thetargeting ligand is exposed and readily available for interaction withthe intended target.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification and a study of the drawings.

SUMMARY OF THE INVENTION

The following aspects and embodiments thereof described and illustratedbelow are meant to be exemplary and illustrative, not limiting in scope.

Accordingly, in one aspect an immunoliposome composition for targetingto a human alpha v integrin subunit is provided. In another aspect, animmunoliposome composition capable of specific binding to a cellexpressing alpha V integrin is provided.

In one aspect, an alpha-V-targeting immunoliposome composition comprisedof liposomes bearing a targeting ligand which is an antibody-derivedconstruct, such as an antibody fragment or derivative, for targeting toa human alpha v integrin subunit is provided.

In one embodiment of the alpha-V-targeting immunoliposome composition ofthe invention, the targeting ligand is comprised of a heavy chainvariable region derived from a parent antibody capable of specificbinding to at least one of alpha-V-beta1, alpha-V-beta3, alpha-V-beta5,alpha-V-beta6, alpha-V-beta8. In a specific embodiment the targetingligand comprises the antibody heavy chain variable region residues 1-119of SEQ ID NO: 1 comprising a framework-1 (FRI), complementaritydetermining region 1 (CDR1), FR2, CDR2, FR3, CDR3 and FR4 sequences. Inone embodiment of the alpha-V-targeting immunoliposome composition ofthe invention, the targeting ligand is comprised of a light chainvariable region residues 1-108 of SEQ ID NO: 2 comprising FRI, CDR1,FR2, CDR2, FR3, CDR3 and FR4 sequences.

In still another aspect, of the alpha-V-targeting immunoliposomecomposition of the invention, the targeting ligand is comprised ofantibody heavy and light chain variable region having a sequenceidentified as SEQ ID NO: 1 residues 1-119 and SEQ ID NO: 2, residues1-108.

In these various embodiment, the alpha-V-targeting immunoliposomeinclude an active entrapped in the liposomes, where ‘entrapped’ intendsassociated with the liposome lipid bilayer or with the internal aqueouscompartments. The agent, in one embodiment, is a therapeutic agent, suchas an antineoplastic agent. In a specific embodiment, the antineoplasticis a cytotoxic or cytostatic agent, such as doxorubicin. In anotheraspect, a method of treating a condition characterized by cells thatexpress one or more of alpha-V-beta1, alpha-V-beta3, alpha-V-beta5,alpha-V-beta6, alpha-V-beta8 is provided. The method includesadministering an alphaV-targeting immunoliposome composition comprisedof a targeting ligand comprising an antibody-derived construct asdescribed above.

In another aspect, a method of treating a condition characterized bycells that express at least one of alpha-V-beta1, alpha-V-beta3,alpha-V-beta5, alpha-V-beta6, and alpha-V-beta8 is provided. The methodcomprises administering immunoliposomes comprised of an isolatedanti-alpha-V subunit monoclonal antibody, the antibody having at leastone variable region having a sequence identified as SEQ ID NO: 1residues 1-119 or SEQ ID NO: 2, residues 1-108.

In yet another aspect, the invention includes a method of treating acondition characterized by cells that express at least one ofalpha-V-beta1, alpha-V-beta3, alpha-V-beta5, alpha-V-beta6, andalpha-V-beta8, comprising administering immunoliposomes comprised of atargeting ligand comprising an antibody-derived construct as describedabove.

In still another aspect, the invention includes a method of treating acondition characterized by cells that express at least one ofalpha-V-beta1, alpha-V-beta3, alpha-V-beta5, alpha-V-beta6, andalpha-V-beta8, comprising administering the alpha-V-targetingimmunoliposome composition comprising a heavy chain variable regioncomprising FRI, CDRI, FR2, CDR2, FR3, CDR3 and FR4 sequences and a lightchain variable region comprising FRI, CDRI, FR2, CDR2, FR3, CDR3 and FR4sequences, wherein: (a) the heavy chain variable region CDR sequencesare selected from those of SEQ ID NO: 1, and conservative modificationsthereof; (b) the light chain variable region CDR sequences are selectedfrom those of SEQ ID NO: 2, and conservative modifications thereof.

In a preferred embodiment, the methods find use in treating a neoplasmcharacterized by cells that express at least one of alpha-V-beta1,alpha-V-beta3, alpha-V-beta5, alpha-V-beta6, and alpha-V-beta8.

In another aspect, a method for inhibiting the proliferation and/orgrowth of a cell expressing alpha-V integrin, and/or inducing killing ofa cell expressing alpha-V integrin is provided, wherein cells arecontacted with (e.g., administering to a subject) an alpha-V-targetingimmunoliposome composition.

Another aspect includes a therapeutic liposome composition sensitized toa target cell, comprising liposomes having an entrapped therapeuticagent, the liposomes including one or more targeting anti-alphaVantibodies in the form of a targeting conjugate. The targeting-ligandconjugate is comprised of (a) a lipid having a polar head group and ahydrophobic tail, (b) a hydrophilic polymer having a proximal end and adistal end, where the polymer is attached at its proximal end to thehead group of the lipid, and (c) an anti-alphaV antibody-derivedconstruct attached to the distal end of the polymer.

Also contemplated is a method of formulating a therapeutic liposomecomposition having sensitivity to a target cell. The method includes thesteps of (i) providing a liposome formulation composed of pre-formedliposomes having an entrapped therapeutic agent; (ii) providing atargeting conjugate composed of (a) a lipid having a polar head groupand a hydrophobic tail, (b) a hydrophilic polymer having a proximal endand a distal end, where the polymer is attached at its proximal end tothe head group of the lipid, and (c) an anti-alpha V antibody targetingligand attached to the distal end of the polymer; (iii) combining theliposome formulation and the targeting conjugate to form thetherapeutic, target-cell sensitive liposome composition. In oneembodiment, combining includes incubating under conditions effective toachieve insertion of the selected targeting conjugate into the liposomesof the selected liposome formulation.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to thedrawings and by study of the following descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the percentage of Fab-PEG-DSPE conjugate remainingin the liposome (diamonds) and dissociated into human plasma (squares),as a function of incubation time, in hours, in human plasma;

FIGS. 2A-2D are images, obtained using a confocal microscope, of A375S.2cells incubated at 4° C. for 30 minutes with liposomes containing afluorescent marker, where FIG. 2A-2B correspond to cells incubated withliposomes lacking a targeting ligand, and FIGS. 2C-2D are images ofcells incubated with liposomes bearing alpha-integrin Fab targetingligands (90:1 Fab:liposome);

FIGS. 3A-3H are images, obtained using a confocal microscope, of A375.S2cells incubated at 37° C. for 10 minutes with liposomes containing afluorescent marker, washed and incubated for 1 hours at 37° C., wherethe images correspond to untreated cells (FIGS. 3A-3B), cells treatedwith free doxorubicin (FIGS. 3C-3D), cells treated with liposomeslacking a targeting ligand (FIGS. 3E-3F), and cells incubated withliposomes bearing alpha-integrin Fab targeting ligands (90:1Fab:liposome, FIGS. 3G-3H);

FIGS. 4A-4J are images, obtained using a confocal microscope, of A375.S2cells incubated at 37° C. for 10 minutes with liposomes containing afluorescent marker, washed and incubated for 0, 6, or 24 hours at 37°C., where the images correspond to untreated cells (FIGS. 4A-4B), cellstreated with liposomes bearing alpha-integrin Fab targeting ligands(90:1 Fab:liposome) and incubated for 0 hours (FIGS. 4C-4D), 6 hours(FIGS. 4E-4F), 24 hours (FIGS. 4G-4H), or with liposomes lacking atargeting ligand (FIGS. 4I-4J; 24 hour post-wash incubation);

FIGS. 5A-5H are images, obtained using a confocal microscope, of B16-F10cells incubated at 37° C. for 10 minutes with liposomes containingdoxorubicin, washed and incubated for 1 hours at 37° C., where theimages correspond to untreated cells (FIGS. 5A-5B), cells treated withfree doxorubicin (FIGS. 5C-5D), cells treated with liposomes lacking atargeting ligand (FIGS. 5E-5F), and cells incubated with liposomesbearing alpha-integrin Fab targeting ligands (90:1 Fab: liposome, FIGS.5G-5H);

FIGS. 6A-6C are graphs showing the percent of viable A375.S2 cells,expressed as a percent of untreated control cells, as a function ofdoxorubicin concentration, in μg/mL, the doxorubicin in free form(squares), entrapped in liposomes lacking a targeting ligand(triangles), entrapped in liposomes bearing alpha-integrin Fab targetingligands at Fab:liposome ratios of 15:1 (x symbols), 40:1 (FIG. 6A,diamonds), 90:1 (FIG. 6B, diamonds; FIGS. 6A-6C, * symbols), 180:1 (FIG.6C, diamonds);

FIGS. 7A-7B are graphs showing the percent of viable MDA-MB-231 cells,expressed as a percent of untreated control cells, as a function ofdoxorubicin concentration, in μg/mL, the doxorubicin in free form(squares), entrapped in liposomes lacking a targeting ligand(triangles), entrapped in liposomes bearing alpha-integrin Fab targetingligands at Fab:liposome ratios of 15:1 (x symbols), 40:1 (FIG. 7A,diamonds; FIG. 7B, circles), and 90:1 (* symbols);

FIG. 8 is a graph showing the percent of viable A2780 cells, expressedas a percent of untreated control cells, as a function of doxorubicinconcentration, in μg/mL, the doxorubicin in free form (squares),entrapped in liposomes lacking a targeting ligand (triangles), entrappedin liposomes bearing alpha-integrin Fab targeting ligands atFab:liposome ratios of 15:1 (diamonds), 40:1 (* symbols), and 90:1(circles);

FIG. 9 is a graph showing the percent of viable B16-F10 cells, expressedas a percent of untreated control cells, as a function of doxorubicinconcentration, in μg/mL, the doxorubicin in free form (squares),entrapped in liposomes lacking a targeting ligand (triangles), orentrapped in liposomes bearing alpha-integrin Fab targeting ligands atFab:liposome ratios of 90:1 (diamonds);

FIG. 10 is a graph showing the doxorubicin concentration, in ng/mL, as afunction of time, in hours, after a single bolus intravenous injectioninto mice of liposomes containing entrapped doxorubicin and lacking atargeting ligand (“S-DOX”, closed circles) or bearing alpha-integrin Fabtargeting ligands at Fab:liposome ratios of 15:1 (open circles), 40:1(open squares), 90:1 (open diamonds), and 180:1 (open triangles);

FIG. 11 is a graph showing the doxorubicin concentration, in ng/mL, as afunction of time, in hours, after a single bolus intravenous injectioninto rats of liposomes containing entrapped doxorubicin and lacking atargeting ligand (“S-DOX”, closed circles) or bearing alpha-integrin Fabtargeting ligands at Fab:liposome ratios of 15:1 (open circles), 30:1(open squares), 60:1 (open diamonds), and 90:1 (open triangles);

FIGS. 12A-12B are graphs showing the relative tumor volume, in percent(FIG. 12A) and relative body weight, in percent (FIG. 12B), as afunction of time, in days, for animals bearing a mammary carcinomaxenograft and left untreated (open squares) or treated with liposomescontaining entrapped doxorubicin at doses of 1 mg/kg and 4 mg/kg, theliposomes lacking a targeting ligand (“S-DOX”, closed and opentriangles) or bearing alpha-integrin Fab targeting ligands atFab:liposome ratios of 15:1 (closed and open circles), 40:1 (closed andopen squares), 90:1 (closed and open diamonds);

FIG. 12C shows the survival of test animals bearing a mammary carcinomaxenograft as a function of time, in days, the animals left untreated(inverted triangles) or treated with liposomes containing entrappeddoxorubicin at doses of 1 mg/kg and 4 mg/kg, the liposomes lacking atargeting ligand (“S-DOX”, closed and open squares) or bearingalpha-integrin Fab targeting ligands at Fab:liposome ratios of 15:1(closed and open diamonds), 40:1 (closed and open triangles), or 90:1(closed and open circles);

FIGS. 13A-13B are graphs showing the mean tumor volume, in mm³, in ratsbearing a human melanoma xenograft, as a function of time, in days,after initiation of treatment with saline, or with doxorubicin a dosesof 2 mg/kg (FIG. 13A) or 0.5 mg/kg (FIG. 13B), the doxorubicin entrappedin liposomes lacking a targeting ligand (“SLD”) or bearingalpha-integrin Fab targeting ligands at Fab:liposome ratios of 15:1 or30:1; and

FIG. 14A-14B are graphs showing the binding of CNTO95 derived scFV toalpha-V-beta3 (A) or alpha-V-beta5 (B) coated plates and detected bybinding of either an anti-idiotype antibody (anti-ids) or and antibodyto the hexahistidine tail.

FIG. 15A-15B are graphs showing a competitive binding of assay betweenCNTO95 derived scFV and the native CNTO95 to alpha-V-beta3 (A) oralpha-V-beta5 (B) coated plates with CNTO95 Fab a positive and anti-her2scFv (F5) a negative control. The detection antibody was HRP anti-humanFc.

FIG. 16 shows a graph of the relationship between cell viability andtreatment with various concentration of doxorubicin in differentcompositions: free doxorubicin, as liposomal doxorubicin (DOXIL), ortargeted liposomal doxorubicin using scFV on the surface of the liposomeat a ratio of targeting ligand: liposome of 15:1, 40:1, and 90:1.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: Description Features 1 Parent antibody: heavy chain 2 Parentantibody: light chain 3 Secreted Fab heavy chain 4 Single Chain antibodyderived from parent antibody 5 Nucleic acid construct for expression ofscFv in E. coli

DETAILED DESCRIPTION I. Definitions & Abbreviations Abbreviations

CV column volume; Fv, antibody variable fragment consisting of VH andVL; scFv, single chain variable fragment; VH, variable heavy; VL,Variable light; PEG, Polyethylene Glycol; Gly4Cys, four glycine residuesfollowed by a cysteine residue; His Tag, six histidine amino acidresidues at the C-terminus of the protein; Fc, Fragment crystallizable

Definitions

The term “alpha-V (αv) integrin”, “alpha-V subunit integrin”, and“alpha-V subunit containing integrin” are used interchangeably herein tomean alpha-V transmembrane glycoprotein subunits of a functionalintegrin heterodimer and include all of the variants, isoforms andspecies homologs of alpha-V. Alpha-V polypeptides include one or moreisoforms of proteins encoded by the ITGAV gene having names integrin,alpha-V (vitronectin receptor, alpha polypeptide, antigen CD51); otheraliases include, CD51, MSK8, VNRA; and other designations are integrin,alpha-V (vitronectin receptor, alpha polypeptide); antigen identified bymonoclonal antibody L230; integrin alpha-V. The gene is located on humanchromosome 2; location: 2q31-q32 (MIM: 193210; GeneID: 3685) Thealpha-V-comprising integrins bind a wide variety of ligands. Humanantibodies of the invention may, in certain cases, cross-react withalpha-V from species other than human, or other proteins that arestructurally related to human alpha-V (e.g., human alpha-V homologs). Inother cases, the antibodies may be completely specific for human alpha-Vand not exhibit species or other types of cross-reactivity.

As used herein, an “antibody” includes whole antibodies and any antigenbinding fragment or single chain fragment thereof. Thus the antibodyincludes any protein or peptide containing molecule that comprises atleast a portion of an immunoglobulin molecule, such as but not limitedto at least one complementarity determining region (CDR) of a heavy orlight chain or a ligand binding portion thereof, a heavy chain or lightchain variable region, a heavy chain or light chain constant region, aframework (FR) region, or any portion thereof, or at least one portionof a binding protein, which can be incorporated into an antibody of thepresent invention. An “alpha-V antibody”, “alpha-V subunit antibody” or“alpha-V integrin antibody” is an antibody that specifically binds thealpha-V subunit of an integrin. The term “antibody” is further intendedto encompass antibodies, digestion fragments, specified portions andvariants thereof, including antibody mimetics or comprising portions ofantibodies that mimic the structure and/or function of an antibody orspecified fragment or portion thereof, including single chain antibodiesand fragments thereof. Functional fragments include antigen-bindingfragments that bind to a mammalian alpha-V subunit. Examples of bindingfragments encompassed within the term “antigen binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH, domains; (ii) a F(ab′)₂ fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH,domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., Nature,341:544-546 (1989)), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (known as single chainFv (scFv); see e.g., Bird et al. Science, 242:423-426 (1988), Huston etal., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988)). Such single chainantibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies.

Such fragments can be produced by enzymatic cleavage, synthetic orrecombinant techniques, as known in the art and/or as described herein.Antibodies can also be produced in a variety of truncated forms usingantibody genes in which one or more stop codons have been introducedupstream of the natural stop site. For example, a combination geneencoding a F(ab′)₂ heavy chain portion can be designed to include DNAsequences encoding the CH₁ domain and/or hinge region of the heavychain. The various portions of antibodies can be joined togetherchemically by conventional techniques, or can be prepared as acontiguous protein using genetic engineering techniques.

A “complementarity determining region” or “CDR” refers to regions ofsomatic hypermutation of the immunoglobulin variable genes which occurafter antigen stimulation during the differentiation of the B lymphocytein the lymph glands leading to an amino acid sequence in the variableregion of an antibody which impart the affinity and specificity ofbinding to the antibody; positioned at the end of several loopedstructures within the variable domain, CDRs form a surface that is“complementary to” the surface of an antigen or an epitope of thatantigen.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Epitopes resulting fromconformational folding of the integrin molecule which arise when aminoacids from differing portions of the linear sequence of the integrinmolecule come together in close proximity in three-dimensional space.Such conformational epitopes are distributed on the extracellular sideof the plasma membrane. Conformational and nonconformational epitopesare distinguished in that the binding to the former but not the latteris lost in the presence of denaturing solvents.

A “framework region” or FR” refers to amino acid sequences which arefound between complementarity determining regions (CDRs) in an antibodyvariable domain and are derived from the germline Heavy chain Variable(IGHV) genes (V, D, J genes) sequences of the human antibody genes.

Unless otherwise noted, the term “incubating” refers to conditions oftime, temperature and liposome lipid composition which allow forpenetration and entry of a selected component, such as a lipid or lipidconjugate, into the lipid bilayer of a liposome.

Unless otherwise noted, the term “pre-formed liposomes” refers tointact, previously formed unilamellar or multilamellar lipid vesicles.

Unless otherwise noted, the term “sensitized to a cell” or “target-cellsensitized” refers to a liposome that includes a ligand or affinitymoiety covalently bound to the liposome and having binding affinity foralpha-V-beta3 (αvβ3) and alpha-V-beta5 (αvβ5) receptor expressed orother alpha-V subunit-containing integrins on a cell.

Unless otherwise noted, the term “therapeutic liposome composition”refers to liposomes that include a therapeutic agent entrapped in theaqueous spaces of the liposomes or in the lipid bilayers of theliposomes.

Unless otherwise noted, the term “vesicle-forming lipid” refers to anylipid capable of forming part of a stable micelle or liposomecomposition and typically including one or two hydrophobic, hydrocarbonchains or a steroid group and may contain a chemically reactive group,such as an amine, acid, ester, aldehyde or alcohol, at its polar headgroup.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences. Thus as used herein, the term “human antibody”refers to an antibody in which substantially every part of the protein(e.g., CDR, framework, C_(L), C_(H) domains (e.g., C_(H)1, C_(H)2,C_(H)3), hinge, (V_(L), V_(H))) is substantially non-immunogenic inhumans, with only minor sequence changes or variations. Similarly,antibodies designated primate (monkey, baboon, chimpanzee, etc.), rodent(mouse, rat, rabbit, guinea pig, hamster, and the like) and othermammals designate such species, sub-genus, genus, sub-family, familyspecific antibodies. Further, chimeric antibodies include anycombination of the above. Such changes or variations optionally andpreferably retain or reduce the immunogenicity in humans or otherspecies relative to non-modified antibodies. Thus, a human antibody isdistinct from a chimeric or humanized antibody. It is pointed out that ahuman antibody can be produced by a non-human animal or prokaryotic oreukaryotic cell that is capable of expressing functionally rearrangedhuman immunoglobulin (e.g., heavy chain and/or light chain) genes.Further, when a human antibody is a single chain antibody, it cancomprise a linker peptide that is not found in native human antibodies.For example, an Fv can comprise a linker peptide, such as two to abouteight glycine or other amino acid residues, which connects the variableregion of the heavy chain and the variable region of the light chain.Such linker peptides are considered to be of human origin.

As used herein, a human antibody is obtained from a system using humanimmunoglobulin sequences, e.g., by immunizing a transgenic mousecarrying human immunoglobulin genes or by screening a humanimmunoglobulin gene library. A human antibody that is “derived from” ahuman germine immunoglobulin sequence can be identified as such bycomparing the amino acid sequence of the human antibody to the aminoacid sequence of human germine immunoglobulins. Human germine antibodyconsensus sequences for various regions and domains of human antibodies;FR1, FR2, FR3, FR4, CH1, hinge1, hinge2, hinge 3, hinge4, CH2, CH3 orfragment thereof are described in Table 2 of, and optionally with atleast one substitution, insertion or deletion as provided in FIGS. 1-42of, PCT WO05/005604 and U.S. Ser. No. 10/872,932 each entirelyincorporated herein by reference. A selected human antibody typically isat least 90% identical in amino acids sequence to an amino acid sequenceencoded by a human germline immunoglobulin gene and contains amino acidresidues that identify the human antibody as being human when comparedto the germline immunoglobulin amino acid sequences of other species(e.g., murine germline sequences). In certain cases, a human antibodymay be at least 95%, or even at least 96%, 97%, 98%, or 99% identical inamino acid sequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, a human antibody derived from aparticular human germline sequence will display no more than ten aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In certain cases, the human antibody maydisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifference from the amino acid sequence encoded by the germlineimmunoglobulin gene.

A monoclonal antibody from a non-human animal, such as a mouse, rat,baboon, or rabbit, may also be used as a parent antibody providing asource of the alpha-V binding regions of the antibody-derivedtargeting-ligand.

The terms “monoclonal antibody” or “parental antibody” as used hereinrefer to a preparation of antibody molecules of single molecularcomposition. A monoclonal antibody displays a single binding specificityand affinity for a particular epitope. Accordingly, the term “humanmonoclonal antibody” refers to antibodies displaying a single bindingspecificity which have variable and constant regions derived from humangermline immunoglobulin sequences. In one embodiment, the humanmonoclonal antibodies are produced by a hybridoma which includes a Bcell obtained from a transgenic nonhuman animal, e.g., a transgenicmouse, having a genome comprising a human heavy chain transgene and alight chain transgene fused to an immortalized cell.

An “isolated antibody,” as used herein, is intended to refer to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities (e.g., an isolated antibody thatspecifically binds to alpha-V is substantially free of antibodies thatspecifically bind antigens other than alpha-V). An isolated antibodythat specifically binds to an epitope, isoform or variant of humanalpha-V may, however, have cross-reactivity to other related antigens,e.g., from other species (e.g., alpha-V species homologs). Moreover, anisolated antibody may be substantially free of other cellular materialand/or chemicals.

As used herein, “specific binding” refers to antibody binding to apredetermined antigen. Typically, the parental antibody binds with adissociation constant (K_(D)) of 10⁻⁷ M or less, and binds to thepredetermined antigen with a K_(D) that is at least twofold less thanits K_(D) for binding to a non-specific antigen (e.g., BSA, casein)other than the predetermined antigen or a closely-related antigen.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen”.

As used herein, K_(D) refers to the dissociation constant, specifically,the antibody K_(D) for a predetermined antigen, and is a measure ofaffinity of the antibody for a specific target. High affinity antibodieshave a K_(D) of 10⁻⁸ M or less, more preferably 10⁻⁹ M or less and evenmore preferably 10⁻¹⁰ M or less, for a predetermined antigen. Thereciprocal of K_(D) is K_(A), the association constant. The term“k_(dis)” or “k₂”, or “k_(d)”, is intended to refer to the dissociationrate of a particular antibody-antigen interaction. The “K_(D)”, is theratio of the rate of dissociation (k₂), also called the “off-rate(k_(off))”, to the rate of association rate (k₁) or “on-rate (k_(on))”.Thus, K_(D) equals k₂/k₁ or k_(off)/k_(on) and is expressed as a molarconcentration (M). It follows that the smaller the K_(D), the strongerthe binding. So a K_(D) of 10⁻⁶ M (or 1 microM) indicates weak bindingcompared to 10⁻⁹ M (or 1 nM).

II. Immunoliposome Composition

In one aspect, an immunoliposome composition is provided, thecomposition comprised of liposomes that include as a targeting ligand anantibody-derived protein which is a monomeric, dimeric or multimericconstruct, having binding specificity for an αv-comprising integrin onthe surface of a cell. The alpha-V targeting-ligand is incorporated intothe liposomes in the form of a lipid-polymer-protein conjugate, alsoreferred to herein as a lipid-polymer-ligand conjugate. As will bedescribed below, the antibody-derived construct has specific affinityfor αv-integrin receptors, and targets the liposomes to cells thatexpress any of the alpha-V-comprising intergrin heterodimers includingbut not limited to αvβ3, αvβ5 and αvβ6 receptors. The following sectionsdescribe the liposome components, including the liposome lipids andtherapeutic agents, preparation of liposomes bearing an anti-alpha-Vtargeting ligand, and methods of using the liposomal composition fortreatment of disorders characterized by cellular expression ofalpha-V-integrins such as αvβ3, αvβ5, and αvβ6 integrin receptors.

A. Liposome Lipid Components

Liposomes suitable for use in the composition of the present inventioninclude those composed primarily of vesicle-forming lipids. Such avesicle-forming lipid is one which can form spontaneously into bilayervesicles in water, as exemplified by the phospholipids, with itshydrophobic moiety in contact with the interior, hydrophobic region ofthe bilayer membrane, and its head group moiety oriented toward theexterior, polar surface of the membrane. Lipids capable of stableincorporation into lipid bilayers, such as cholesterol and its variousanalogs, can also be used in the liposomes.

The vesicle-forming lipids are preferably lipids having two hydrocarbonchains, typically acyl chains, and a head group, either polar ornonpolar. There are a variety of synthetic vesicle-forming lipids andnaturally-occurring vesicle-forming lipids, including the phospholipids,such as phosphatidylcholine, phosphatidylethanolamine, phosphatidicacid, phosphatidylinositol, and sphingomyelin, where the two hydrocarbonchains are typically between about 14-22 carbon atoms in length, andhave varying degrees of unsaturation. The above-described lipids andphospholipids whose carbon chains have varying degrees of saturation canbe obtained commercially or prepared according to published methods.Other suitable lipids include glycolipids, cerebrosides and sterols,such as cholesterol.

Cationic lipids are also suitable for use in the liposomes of theinvention, where the cationic lipid can be included as a minor componentof the lipid composition or as a major or sole component. Such cationiclipids typically have a lipophilic moiety, such as a sterol, an acyl ordiacyl chain, and where the lipid has an overall net positive charge.Preferably, the head group of the lipid carries the positive charge.Exemplary cationic lipids include 1,2-dioleyloxy-3-(trimethylamino)propane (DOTAP);N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammoniumbromide (DMRIE); N-[1-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE);N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA);3[N-(N′,N′-dimethylaminoethane) carbamoyl]cholesterol (DC-Chol); anddimethyldioctadecylammonium (DDAB). The cationic vesicle-forming lipidmay also be a neutral lipid, such as dioleoylphosphatidyl ethanolamine(DOPE) or an amphipathic lipid, such as a phospholipid, derivatized witha cationic lipid, such as polylysine or other polyamine lipids. Forexample, the neutral lipid (DOPE) can be derivatized with polylysine toform a cationic lipid.

The vesicle-forming lipid can be selected to achieve a specified degreeof fluidity or rigidity, to control the stability of the liposome inserum, to control the conditions effective for insertion of thetargeting conjugate, as will be described, and/or to control the rate ofrelease of the entrapped agent in the liposome. Liposomes having a morerigid lipid bilayer, or a liquid crystalline bilayer, are achieved byincorporation of a relatively rigid lipid, e.g., a lipid having arelatively high phase transition temperature, e.g., up to 60° C. Rigid,i.e., saturated, lipids contribute to greater membrane rigidity in thelipid bilayer. Other lipid components, such as cholesterol, are alsoknown to contribute to membrane rigidity in lipid bilayer structures.

On the other hand, lipid fluidity is achieved by incorporation of arelatively fluid lipid, typically one having a lipid phase with arelatively low liquid to liquid-crystalline phase transitiontemperature, e.g., at or below room temperature.

The liposomes also include a vesicle-forming lipid derivatized with ahydrophilic polymer. As has been described, for example in U.S. Pat. No.5,013,556, including such a derivatized lipid in the liposomecomposition forms a surface coating of hydrophilic polymer chains aroundthe liposome. The surface coating of hydrophilic polymer chains iseffective to increase the in vivo blood circulation lifetime of theliposomes when compared to liposomes lacking such a coating.

Vesicle-forming lipids suitable for derivatization with a hydrophilicpolymer include any of those lipids listed above, and, in particularphospholipids, such as distearoyl phosphatidylethanolamine (DSPE).

Hydrophilic polymers suitable for derivatization with a vesicle-forminglipid include polyvinylpyrrolidone, polyvinylmethylether,polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline,polyhydroxypropylmethacrylamide, polymethacrylamide,polydimethylacrylamide, polyhydroxypropylmethacrylate,polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose,polyethyleneglycol, polyaspartamide and hydrophilic peptide sequences.The polymers may be employed as homopolymers or as block or randomcopolymers.

A preferred hydrophilic polymer chain is polyethyleneglycol (PEG),preferably as a PEG chain having a molecular weight between 500-10,000daltons, more preferably between 750-10,000 daltons, still morepreferably between 750-5000 daltons. Methoxy or ethoxy-capped analoguesof PEG are also preferred hydrophilic polymers, commercially availablein a variety of polymer sizes, e.g., 120-20,000 Daltons.

Preparation of vesicle-forming lipids derivatized with hydrophilicpolymers has been described, for example in U.S. Pat. No. 5,395,619.Preparation of liposomes including such derivatized lipids has also beendescribed, where typically between 1-20 mole percent of such aderivatized lipid is included in the liposome formulation (see, forexample, U.S. Pat. No. 5,013,556).

B. Anti-Alpha-V Targeting Ligand

The antibody derived targeting ligand of the invention, as definedherein, to be used to prepare the liposomal compositions of the presentinvention, may be derived from any anti-alpha-V specific antibody orselected from a library of pre-formed antibody-derived structures, e.g.a phage library comprising antibody Fab′ or scFv or Fv. In oneembodiment, the antibody for use in the liposome composition describedherein comprises antigen binding domains derived from a humananti-alpha-V antibody generated by immunization of a transgenic mousecontaining genes for the expression of human immunoglobulins.Preparation of a parent anti-alpha-V antibody known as CNTO 95, fromwhich the antigen binding domains are derived is described inPreparation of the antibody is described in detail in PCT publicationno. WO 02/12501 and U.S. Pat. No. 7,163,681 both incorporated byreference herein.

The antibody-derived targeting ligand includes any protein or peptidecontaining molecule that comprises at least a portion of acomplementarity determining region (CDR) of a heavy or light chain or aligand binding portion thereof derived from the antibody designated“CNTO 95” (see PCT publication no. WO 02/12501 and U.S. Publication No.2003/040044), in combination with a heavy chain or light chain variableregion, a heavy chain or light chain constant region, a frameworkregion, or any portion thereof, that can be incorporated into anantibody.

Preferably, the CDR1, 2, and/or 3 of the engineered targeting liganddescribed above comprise the exact amino acid sequence(s) as those ofthe fully human Mab designated CNTO 95, Gen0101, CNTO 95, C371Agenerated by immunization of a transgenic mouse as disclosed herein.However, the ordinarily skilled artisan will appreciate that somedeviation from the exact CDR sequences of CNTO 95 may be possible whilestill retaining the ability of the antibody to bind alpha-V effectively(e.g., conservative substitutions). In a particular embodiment, theantibody or antigen-binding fragment can have an antigen-binding regionthat comprises at least a portion of at least one heavy chain CDR (i.e.,CDR1, CDR2 and/or CDR3) having the amino acid sequence of thecorresponding CDRs 1, 2 and/or 3 (as shown in SEQ ID NO: 1). In anotherparticular embodiment, the antibody or antigen-binding portion orvariant can have an antigen-binding region that comprises at least aportion of at least one light chain CDR (i.e., CDR1, CDR2 and/or CDR3)having the amino acid sequence of the corresponding CDRs 1, 2 and/or 3(as shown in SEQ ID NO: 2) of the light chain of CNTO95. In a preferredembodiment the three heavy chain CDRs and the three light chain CDRs ofthe antibody or antigen-binding fragment have the amino acid sequence ofthe corresponding CDR of mAb CNTO 95 (as shown in SEQ ID Nos: 1 and 2).Accordingly, in another embodiment, the engineered antibody may becomposed of one or more CDRs that are, for example, 90%, 95%, 98% or99.5% identical to one or more CDRs of CNTO 95. Anti-alpha-V subunitantibodies of the present invention can include, but are not limited to,at least one portion, sequence or combination selected from 5 to all ofthe contiguous amino acids of at least one of six CDRs shown in SEQ IDNOS: 1 and 2. An anti-alpha-V subunit antibody can further optionallycomprise a polypeptide of at least one of 70-100% of the contiguousamino acids of at least one of SEQ ID NOS: 1 and 2. For example, theamino acid sequence of a light chain variable region can be comparedwith the sequence of SEQ ID NO: 2, residues 1-108, or the amino acidsequence of a heavy chain CDR3 can be compared with SEQ ID NO: 1,residues 1-119.

As disclosed and claimed herein, the sequences set forth in SEQ ID NOs.1-4 include “conservative sequence modifications”, i.e. amino acidsequence modifications which do not significantly affect or alter thebinding characteristics of the antibody encoded by the nucleotidesequence or containing the amino acid sequence. Such conservativesequence modifications include amino acid substitutions, additions anddeletions. Modifications can be introduced into SEQ ID NOs: 1-2 or tothe nucleic acids encoding them by standard techniques known in the art,such as site-directed mutagenesis and PCR-mediated mutagenesis.Conservative amino acid substitutions include ones in which the aminoacid residue is replaced with an amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a human anti-alpha-Vantibody is preferably replaced with another amino acid residue from thesame side chain family.

In another aspect of the invention, the structural features of a humananti-alpha-V antibody are used to create structurally related a humananti-alpha-V targeting ligand that retain ability to bind to alphaV.More specifically, one or more antigen binding regions, specifically thevariable regions and the CDR regions of the anti-alpha-V antibody can becombined recombinantly with other known human constant regions orframework regions and CDRs to create additional,recombinantly-engineered, human anti-alpha-V targeting moities of theinvention.

At least one antibody of the invention binds at least one specifiedepitope specific to at least one alpha-V subunit protein, subunit,fragment, portion or any combination thereof. The at least one epitopecan comprise at least one portion of said protein, preferably comprisedof at least one extracellular, soluble, external or cytoplasmic portionof said protein. The at least one specified epitope can comprise anycombination of at least one amino acid sequence of at least 1-3 aminoacids to the entire specified portion of contiguous amino acids of aprotein encoded by the ITGAV gene (Gene ID: 3683).

Amino acids in an anti-alpha-V antibody to be used in the presentinvention that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells,Science 244:1081-1085 (1989)). The latter procedure introduces singlealanine mutations at every residue in the molecule. The resulting mutantmolecules are then tested for biological activity, such as, but notlimited to at least one alpha-V subunit neutralizing activity. Sitesthat are critical for antibody binding can also be identified bystructural analysis such as crystallization, nuclear magnetic resonanceor photoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904(1992) and de Vos, et al., Science 255:306-312 (1992)).

The present invention is not limited to the use of CNTO 95 mAb, itsvariable domains, or CDR sequences. It is anticipated that anyappropriate anti-alpha-V antibody and corresponding anti-αv CDRsdescribed in the art may be substituted therefor. Other anti-αv subunitantibodies may be developed by screening hybridomas, combinatoriallibraries, or specific antibody phage display libraries [W. D. Huse etal., 1988, Science, 246:1275-1281] for binding to a human αV-containingintegrin epitope. A collection of antibodies, including hybridomaproducts or antibodies derived from any species immunoglobulinrepertoire may be screened in a conventional competition assay, with oneor more of the known anti-alphaV antibodies described herein. Thus, theinvention may provide an antibody, other than CNTO95 derived antibodies,which is capable of binding to the αv-containing receptors.

In another embodiment, the anti-alpha-V antibody may be 17E6, afragment, or variant thereof based on the binding domains of 17E6 asdescribed in U.S. Pat. No. 5,985,278 which reacts with the αV-chain ofhuman αV-integrins, blocking the attachment to the integrin substrate ofthe αV-integrin bearing cell, triggering reversal of established cellmatrix interaction caused by αV-integrins, blocking tumor development,and showing no cytotoxic activity. In yet another embodiment, theanti-alpha-V antibody may be murine monoclonal B9 and the humanizedantibody HuB9 as described in U.S. Pat. No. 6,160,099 which react withthe αV-chain of human α_(v)β₃ and α_(v)β₅ integrins.

Variations derived from the naturally occurring antibody structure, asdefined herein which are particularly useful in the present inventioninclude Fabs and scFv. ScFv (single-chain variable fragment antibody) isa minimal antibody moiety in which the variable regions from the heavyand light chains (Vh and Vl) of immunoglobulin are joined by a flexiblelinker (U.S. Pat. No. 5,260,203). The resulting linked domains representa variable region fragment, which retains both affinity and specificityof the parent antibody. These small antibody fragments can be producedin E. coli providing a fast and economic manufacturing option. A scFv ofCNTO95 was developed as a targeting moiety to specifically direct drugcontaining STEALTH liposomes to αVb3 and αVb5 integrins which are knownto be present on numerous types of cancer cells as well as angiogenicendothelial cells thereby representing an ideal targeting opportunityfor drug delivery to subjects with neoplastic disease. One particularadvantage of the scFv is that, in contrast to larger antibody fragments,a scFv contains only 4 cysteine residues and these are engaged in the 2disulfide bonds of the V-domains. This facilitates introduction of afree cysteine residue for chemical conjugation. Moreover, the small sizeof the scFv is less likely to impact the stability and low non-specificinteractions of STEALTH Liposomes. A further advantage of an scFv withthe alpha-V targeting properties of CNTO95 are the ability to causereceptor internalization upon binding. Certain specific embodiments ofthe anti-alpha-V targeting antibody constructs are single chain bindingfragments (scFv) which may be prepared from a parent antibody asdescribed in Example 11 and as exemplified by SEQ ID NO: 4.

In another embodiment the targeting antibody is a Fab, which representsa monovalent binding fragment of an antibody, comprising both heavychain and light chain portions of an antibody, which may be produced bycleavage from an antibody or be synthesized recombinantly and expressedas the heterodimeric structure. In the present invention, exemplaryforms of Fabs produced by both processes are described in Example 2 and9. A Fab derived from cleavage of the parent CNTO95 IgG comprising thefull-length heavy and light chains of the antibody (SEQ ID NO: 1 and 2,respectively) cleaved by pepsin is represented by residues 1-234 or SEQID NO: 1 and the full-length light chain (SEQ ID NO: 2). A recombinantlyengineered host cell line expressing and secreting a Fab (sFab) which isrepresented by SEQ ID NO: 3 and SEQ ID NO: 2 is particularly useful forthe purposes of conjugation and insertion into a pre-formed liposomeamong other uses.

It is useful for the targeting antibody of the present invention tocomprise a predetermined site for conjugation to a chemically moietycapable of insertion into the lipid structure of the liposome. Whilechemical modification of and addition of reactive groups is possible bystandard techniques, it is convenient to genetically encode such a siteinto the structure of the antibody whenever possible. In the case of therecombinantly expressed and secreted antibody targeting constructs,including the sFab and scFv antibody constructs, each polypeptide chainhas an additional C-terminal tail amino acid sequence having a means forchemically modifying the polypeptide such as through a free sulfhydrylof a cysteine side chain or an amine residue of a lysine sidechain.Exemplary methods of incorporating a predetermined site for conjugationare taught in e.g. U.S. Pat. No. 5,837,846 which is incorporated hereinby reference and which embodiments include a C-terminal cysteine or aC-terminal tail peptide bonded to the C-terminus of the antibody heavychain or heavy chain fragment or scFv and, optionally, having an aminoacid sequence selected from the group consisting of Ser-Cys, (Gly)₄-Cys,and (His)₆-(Gly)₄-Cys thereby incorporating linking means as well aspurification means (his-tag).

C. Preparation of Lipid-Polymer-Antibody Conjugate

The anti-alpha-V antibody is covalently attached to the free distal endof a hydrophilic polymer chain, which is attached at its proximal end toa vesicle-forming lipid. There are a wide variety of techniques forattaching a selected hydrophilic polymer to a selected lipid andactivating the free, unattached end of the polymer for reaction with aselected ligand, and in particular, the hydrophilic polymerpolyethyleneglycol (PEG) has been widely studied (Allen, T. M., et al.,Biochemicia et Biophysica Acta, 1237:99-108 (1995); Zalipsky, S.,Bioconjugate Chem., 4(4):296-299 (1993); Zalipsky, S., et al. FEBSLett., 353:71-74 (1994); Zalipsky, S. et al., Bioconjugate Chemistry,6(6):705-708 (1995); Zalipsky, S., in STEALTH LIPOSOMES (D. Lasic and F.Martin, Eds.) Chapter 9, CRC Press, Boca Raton, Fla. (1995)).

Generally, the PEG chains are functionalized to contain reactive groupssuitable for coupling with, for example, sulfhydryls, amino groups, andaldehydes or ketones (typically derived from mild oxidation ofcarbohydrate portions of an antibody) present in a wide variety ofligands. Examples of such PEG-terminal reactive groups include maleimide(for reaction with sulfhydryl groups), N-hydroxysuccinimide (NHS) orNHS-carbonate ester (for reaction with primary amines), hydrazide orhydrazine (for reaction with aldehydes or ketones), iodoacetyl(preferentially reactive with sulfhydryl groups) and dithiopyridine(thiol-reactive). Synthetic reaction schemes for activating PEG withsuch groups are set forth in U.S. Pat. Nos. 5,631,018, 5,527,528,5,395,619, and the relevant sections describing synthetic reactionprocedures are expressly incorporated herein by reference.

In supporting studies, the anti-integrin antibody fragment was a Fab′antibody produced by enzymatic cleavage of a full length parentantibody, which was attached to a lipid-PEG conjugate, as described inExample 2. In brief, a lipopolymer with a reactive end,maleimide-PEG-DSPE, was inserted into drug loaded liposomes, forsubsequence conjugation between the reactive PEG end and the Fab′targeting ligand. The Fab′ was prepared by first reducing F(ab′)₂ tocleave solvent accessible disulfide bonds and then reoxidizing theprotein in a controlled manner to selectively reform the disulfide bondsbetween the heavy and light chains, thus forming Fab′ at a high purity.The reoxidized Fab′ was then added to the liposomes bearing reactivemaleimide groups to conjugate the Fab′ ligand to the external surface ofthe liposomes.

In another study, as described in Example 9, the anti-alpha-Vantibody-derived construct was also a Fab′ fragment but was a variant ofthe parental sequence (SEQ ID NO: 3) synthesized by recombinant methodsand conjugated to a PEGylated-lipid for surface insertion into apre-formed liposome.

In another study, as described in Example 11, the anti-alpha-Vantibody-derived construct was a scFv which was a produced variant ofthe parental sequence heavy chain (SEQ ID NO: 1) variable domain withthe parental sequence light chain (SEQ ID NO: 2) variable domain with aflexible polypeptide linker interposed therebetween. The construct wasproduced by linking coding sequences for the variable domains operablywith a coding sequence for the linking a sequence by recombinantmethods. The expressed purified scFv, which retained binding specificityfor alphaV-integrins was conjugated to a PEGylated-lipid for surfaceinsertion into a pre-formed liposome.

D. Liposome Preparation

Various approaches have been described for preparing liposomes having atargeting ligand attached to the distal end of liposome-attached polymerchains. One approach involves preparation of lipid vesicles whichinclude an end-functionalized lipid-polymer derivative; that is, alipid-polymer conjugate where the free polymer end is reactive or“activated” (see, for example, U.S. Pat. Nos. 6,326,353 and 6,132,763).Such an activated conjugate is included in the liposome composition andthe activated polymer ends are reacted with a targeting ligand afterliposome formation. Example 2 describes preparation of liposomes usingthis approach.

In another approach, the lipid-polymer-ligand conjugate is included inthe lipid composition at the time of liposome formation (see, forexample, U.S. Pat. Nos. 6,224,903, 5,620,689).

In another method of preparing a targeted liposome, a micellar solutionof the lipid-polymer-ligand conjugate is incubated with a suspension ofliposomes and the lipid-polymer-ligand conjugate is inserted into thepre-formed liposomes (see, for example, U.S. Pat. Nos. 6,056,973 and6,316,024). Examples 3, 9 and 11 describe preparation of liposomes usingthis approach.

It will be appreciated that liposomes carrying an entrapped agent andbearing surface-bound targeting ligands, i.e., targeted, therapeuticliposomes, are prepared by any of these approaches. A preferred methodof preparation is the insertion method, where pre-formed liposomes andare incubated with the targeting conjugate to achieve insertion of thetargeting conjugate into the liposomal bilayers. In this approach,liposomes are prepared by a variety of techniques, such as thosedetailed in Szoka, F., Jr., et al., Ann. Rev. Biophys. Bioeng., 9:467(1980), and specific examples of liposomes prepared in support of thepresent invention will be described below. Typically, the liposomes aremultilamellar vesicles (MLVs), which can be formed by simple lipid-filmhydration techniques. In this procedure, a mixture of liposome-forminglipids of the type detailed above dissolved in a suitable organicsolvent is evaporated in a vessel to form a thin film, which is thencovered by an aqueous medium. The lipid film hydrates to form MLVs,typically with sizes between about 0.1 to 10 microns.

The liposomes can include a vesicle-forming lipid derivatized with ahydrophilic polymer to form a surface coating of hydrophilic polymerchains on the liposomes surface. Addition of a lipid-polymer conjugateis optional, since after the insertion step the liposomes will includelipid-polymer-targeting ligand. Additional polymer chains added to thelipid mixture at the time of liposome formation and in the form of alipid-polymer conjugate result in polymer chains extending from both theinner and outer surfaces of the liposomal lipid bilayers. Addition of alipid-polymer conjugate at the time of liposome formation is typicallyachieved by including between 1-20 mole percent of thepolymer-derivatized lipid with the remaining liposome formingcomponents, e.g., vesicle-forming lipids. Exemplary methods of preparingpolymer-derivatized lipids and of forming polymer-coated liposomes havebeen described in U.S. Pat. Nos. 5,013,556, 5,631,018 and 5,395,619,which are incorporated herein by reference. It will be appreciated thatthe hydrophilic polymer may be stably coupled to the lipid, or coupledthrough an unstable linkage, which allows the coated liposomes to shedthe coating of polymer chains as they circulate in the bloodstream or inresponse to a stimulus.

The liposomes also include a therapeutic or diagnostic agent, andexemplary agents are provided below. The selected agent is incorporatedinto liposomes by standard methods, including (i) passive entrapment ofa water-soluble compound by hydrating a lipid film with an aqueoussolution of the agent, (ii) passive entrapment of a lipophilic compoundby hydrating a lipid film containing the agent, and (iii) loading anionizable drug against an inside/outside or outside/inside liposomechemical or pH gradient. Other methods, such as reverse-phaseevaporation, are also suitable.

After liposome formation, the liposomes can be sized to obtain apopulation of liposomes having a substantially homogeneous size range,typically between about 0.01 to 0.5 microns, more preferably between0.03-0.40 microns. One effective sizing method for REVs and MLVsinvolves extruding an aqueous suspension of the liposomes through aseries of polycarbonate membranes having a selected uniform pore size inthe range of 0.03 to 0.2 micron, typically 0.05, 0.08, 0.1, or 0.2microns. The pore size of the membrane corresponds roughly to thelargest sizes of liposomes produced by extrusion through that membrane,particularly where the preparation is extruded two or more times throughthe same membrane. Homogenization methods are also useful fordown-sizing liposomes to sizes of 100 nm or less (Martin, F. J., inSPECIALIZED DRUG DELIVERY SYSTEMS—MANUFACTURING AND PRODUCTIONTECHNOLOGY, P. Tyle, Ed., Marcel Dekker, New York, pp. 267-316 (1990)).

In one embodiment, after formation of the liposomes, a targeting ligandis incorporated to achieve a target-cell sensitized, therapeuticliposome. The targeting ligand can be incorporated attaching the ligandto an activated end on the hydrophilic polymer chain (Example 2) or byincubating the pre-formed liposomes with the lipid-polymer-ligandconjugate (Examples 3, 9, and 11). In the latter approach, thepre-formed liposomes and the conjugate are incubated under conditionseffective to achieve insertion of the conjugate into the liposomebilayer. More specifically, the two components are incubated togetherunder conditions which achieve insertion of the conjugate in such a waythat the targeting ligand is oriented outwardly from the liposomesurface, and therefore available for interaction with its cognatereceptor. It will be appreciated that the conditions effective toachieve insertion of the targeting conjugate into the liposome aredetermined based on several variables, including, the desired rate ofinsertion, where a higher incubation temperature may achieve a fasterrate of insertion, the temperature to which the ligand can be safelyheated without affecting its activity, and to a lesser degree the phasetransition temperature of the lipids and the lipid composition. It willalso be appreciated that insertion can be varied by the presence ofsolvents, such as amphipathic solvents including polyethyleneglycol andethanol, or detergents.

The targeting conjugate, in the form of a lipid-polymer-ligandconjugate, will typically form a solution of micelles when the conjugateis mixed with an aqueous solvent. The micellar solution of theconjugates is mixed with a suspension of pre-formed liposomes forinsertion of the conjugate into the liposomal lipid bilayers.Accordingly, in another aspect, a plurality of targeting conjugates,such as a micellar solution of targeting conjugates, for use inpreparing a targeted, therapeutic liposome composition, is contemplated.Each conjugate is composed of (i) a lipid having a polar head group anda hydrophobic tail, (ii) a hydrophilic polymer having a proximal end anda distal end, where the polymer is attached at its proximal end to thehead group of the lipid, and (iii) an anti-alpha-V antibody targetingligand attached to the distal end of the polymer.

Also contemplated is a method of formulating a therapeutic liposomecomposition having sensitivity to a target cell. The method includes thesteps of (i) providing a liposome formulation composed of pre-formedliposomes having an entrapped therapeutic agent; (ii) providing atargeting conjugate composed of (a) a lipid having a polar head groupand a hydrophobic tail, (b) a hydrophilic polymer having a proximal endand a distal end, where the polymer is attached at its proximal end tothe head group of the lipid, and (c) an anti-alpha-V antibody targetingligand attached to the distal end of the polymer; (iii) combining theliposome formulation and the targeting conjugate to form thetherapeutic, target-cell sensitive liposome composition. In oneembodiment, combining includes incubating under conditions effective toachieve insertion of the selected targeting conjugate into the liposomesof the selected liposome formulation.

Exemplary Immunoliposomes

In supporting studies, immunoliposomes having an anti-αv integrin Fabantibody were prepared as described in Example 1 and 2 and with analternative embodiment of a Fab secreted by an engineered host cell, inExample 9. In another embodiment, immunoliposomes having an anti-αvintegrin scFv targeting moiety were prepared as described in Example 12.In brief, liposomes were prepared from the lipids HSPC, cholesterol. Thetherapeutic agent doxorubicin was loaded into the liposomes by remoteloading against an ammonium ion gradient (Doxil®). In one method ofattaching the targeting moiety to the liposome, an anti-αV Fab having afree sulfhydryl near the C-terminus was attached to the active end ofthe PEG chains previously inserted as Mal-PEG-DSPE. Liposomeformulations having various antibody:liposome ratios were prepared. Inan alternate method of attaching an anti-αV Fab, a Fab having a freesulfhydryl near the C-terminus can be conjugated to the Mal-PEG-DSPE andthe Fab-PEG-DSPE conjugate inserted into pre-formed liposomes as taughtin Example 3 and Example 9. While Example 12 is directed to a scFv thatis conjugated to a post MalPEG-DSPE inserted liposome, other scantibodies exist that do not denature in insertion conditions and mayalso be inserted into pre-formed, preloaded liposomes at various ligandto liposome ratios.

In certain embodiments the alphaV-targeted liposome of the inventiondescribed in the examples set forth below, the alpha-V -targetedimmunoliposomes were characterized, in vitro and in certain examples, invivo.

III. Methods of Use

The liposomes can include a therapeutic or diagnostic agent in entrappedform. Entrapped is intended to include encapsulation of an agent in theaqueous core and aqueous spaces of liposomes as well as entrapment of anagent in the lipid bilayer(s) of the liposomes. Agents contemplated foruse in the composition of the invention are widely varied, and examplesof agents suitable for therapeutic and diagnostic applications are givenbelow.

The targeting ligand included in the liposomes serves to direct theliposomes to a region, tissue, or cell bearing αvβ3, αvβ5 integrin, orother αv-subunit containing integrin receptors. Targeting the liposomesto such a region achieves site specific delivery of the entrapped agent.Disease states having a strong αvβ3, αvβ5 vascular disorders orosteoporosis (αvβ3); tumor angiogenesis, tumor metastasis, tumor growth,multiple sclerosis, neurological disorders, asthma, vascular injury ordiabetic retinopathy (αvβ3 or αvβ5); and, angiogenesis (both αvβ3 andαvβ5).

Additionally, αvβ3 inhibitors or agents which block ligand binding tothe receptor have been found to be useful in treating diseasescharacterized by excessive or inappropriate angiogenesis (i.e. formationof new blood vessels) and inhibiting neoplastic growth and tumormetastasis. Consequently the delivery of an appropriate therapeuticagent to would be expected to enhance this effect.

Moreover, the growth of tumors depends on an adequate blood supply,which in turn is dependent on the growth of new vessels into the tumor;thus, inhibition of angiogenesis can cause tumor regression in animalmodels (Harrison's Principles of Internal Medicine, 1991, 12th ed.).Therefore, an αv-subunit containing integrin-targeted liposomecontaining a therapeutic agent, which inhibit angiogenesis can be usefulin the treatment of cancer by inhibiting tumor growth (Brooks et al.,Cell, 79:1157-1164 (1994)). Evidence has also been presented suggestingthat angiogenesis is a central factor in the initiation and persistenceof arthritic disease and that the vascular integrin αvβ3 may be apreferred target in inflammatory arthritis. Therefore, αvβ3 targetedliposomes that deliver an anti-angiogenesis or appropriate therapeuticdrug to treat arthritis may represent a novel therapeutic approach tothe treatment of arthritic disease, such as rheumatoid arthritis (C. M.Storgard et al., J. Clin. Invest., 103:47-54 (1999)).

Inhibition of the αvβ5 integrin receptor can also preventneovascularization. A monoclonal antibody for αvβ5 has been shown toinhibit VEGF-induced angiogenesis in rabbit cornea and the chickchorioallantoic membrane model (M. C. Friedlander et al., Science,270:1500-1502 (1995)). Thus, anti-alpha-V targeted liposomes, which willnaturally target αvβ5, containing an appropriate therapeutic agent wouldbe useful for treating and preventing macular degeneration, diabeticretinopathy, cancer, and metastatic tumor growth.

Inhibition of αβ integrin receptors can also prevent angiogenesis andinflammation by acting as antagonists of alpha-V-subunit integrinscomprising other β subunits, such as αvβ6 and αvβ8 (MelpoChristofidou-Solomidou et al., American Journal of Pathology, 151:975-83(1997); Xiao-Zhu Huang et al., Journal of Cell Biology, 133:921-28(1996)), again suggesting in disease states where angiogenesis orinflammation is to be treated that αvβ6 targeted liposome containing anappropriate therapeutic agent would provide a novel therapy.

More generally, the anti-alpha-V subunit antibodies or specifiedvariants thereof can be used to measure or effect in an cell, tissue,organ or animal (including mammals and humans), to diagnose, monitor,modulate, treat, alleviate, help prevent the incidence of, or reduce thesymptoms of, at least one condition mediated, affected or modulated byalpha-V integrins. Such conditions are selected from, but not limitedto, diseases or conditions mediated by cell adhesion and/orangiogenesis. Such diseases or conditions include an immune disorder ordisease, a cardiovascular disorder or disease, an infectious, malignant,and/or neurologic disorder or disease, or other known or specifiedalpha-V integrin subunit related conditions. In particular, theantibodies are useful for the treatment of diseases that involveangiogenesis such as disease of the eye and neoplastic disease, tissueremodeling such as restenosis, and proliferation of certain cells typesparticularly epithelial and squamous cell carcinomas. Particularindications include use in the treatment of atherosclerosis, restenosis,cancer metastasis, rheumatoid arthritis, diabetic retinopathy andmacular degeneration. The neutralizing antibodies of the invention arealso useful to prevent or treat unwanted bone resorption or degradation,for example as found in osteoporosis or resulting from PTHrPoverexpression by some tumors. The antibodies may also be useful in thetreatment of various fibrotic diseases such as idiopathic pulmonaryfibrosis, diabetic nephropathy, hepatitis, and cirrhosis.

Thus, in one embodiment, the present invention provides a method formodulating or treating at least one alpha-V subunit related disease, ina cell, tissue, organ, animal, or patient, as known in the art or asdescribed herein, using at least one alpha-V subunit antibody of thepresent invention. One preferred indication are malignant diseases in acell, tissue, organ, animal or patient. Malignant diseases include, butare not limited to, at least one of: leukemia, acute leukemia, acutelymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloidleukemia (AML), chromic myelocytic leukemia (CML), chronic lymphocyticleukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), alymphoma, Hodgkin's disease, a malignant lymphoma, non-hodgkin'slymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma,colorectal carcinoma, pancreatic carcinoma, renal cell carcinoma, breastcancer, nasopharyngeal carcinoma, malignant histiocytosis,paraneoplastic syndrome/hypercalcemia of malignancy, solid tumors,adenocarcinomas, squamous cell carcinomas, sarcomas, malignant melanoma,particularly metastatic melanoma, hemangioma, metastatic disease, cancerrelated bone resorption, cancer related bone pain, and the like.

The immunoliposome includes an agent entrapped within the liposome. Theagent is entrapped in either or both of the aqueous spaces and/or thelipid bilayers. The agent is an active, typically a therapeutic agent,which includes natural and synthetic compounds having the followingtherapeutic activities including but not limited to: steroids,immunosuppressants, antihistamines, non-steroidal anti-asthmatics,non-steroidal anti-inflammatory agents, cyclooxygenase-2 inhibitors,cytotoxic agents, gene therapy agents, radiotherapy agents, and agentscapable of gene knockdown. Imaging agents may also be used in thetargeted liposomes particularly with regard to diagnosis or imaging ofpatients who have cells and tissues sensitized to alpha-V-targetedliposomes.

Examples of these compounds include (a) steroids such as beclomethasone,methylprednisolone, betamethasone, prednisone, dexamethasone, andhydrocortisone; (b) immunosuppressants such as FK-506 typeimmunosuppressants; (c) antihistamines (H1-histamine antagonists) suchas bromopheniramine, chlorpheniramine, dexchlorpheniramine,triprolidine, clemastine, diphenhydramine, diphenylpyraline,tripelennamine, hydroxyzine, methdilazine, promethazine, trimeprazine,azatadine, cyproheptadine, antazoline, pheniramine pyrilamine,astemizole, terfenadine, loratadine, cetirizine, fexofenadine,descarboethoxyloratadine, and the like; (d) non-steroidalanti-asthmatics such as beta2-agonists (terbutaline, metaproterenol,fenoterol, isoetharine, albuterol, bitolterol, salmeterol andpirbuterol), theophylline, cromolyn sodium, atropine, ipratropiumbromide, leukotriene antagonists (zafirlukast, montelukast, pranlukast,iralukast, pobilukast, SKB-106,203), leukotriene biosynthesis inhibitors(zileuton, BAY-1005); (e) non-steroidal antiinflammatory agents (NSAIDs)such as propionic acid derivatives (alminoprofen, benoxaprofen, bucloxicacid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen,ibuprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin,pirprofen, pranoprofen, suprofen, tiaprofenic acid, and tioxaprofen),acetic acid derivatives (indomethacin, acemetacin, alclofenac, clidanac,diclofenac, fenclofenac, fenclozic acid, fentiazac, furofenac, ibufenac,isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin, andzomepirac), fenamic acid derivatives (flufenamic acid, meclofenamicacid, mefenamic acid, niflumic acid and tolfenamic acid),biphenylcarboxylic acid derivatives (diflunisal and flufenisal), oxicams(isoxicam, piroxicam, sudoxicam and tenoxican), salicylates (acetylsalicylic acid, sulfasalazine) and the pyrazolones (apazone,bezpiperylon, feprazone, mofebutazone, oxyphenbutazone, phenylbutazone);(f) cyclooxygenase-2 (COX-2) inhibitors such as celecoxib, rofecoxib,and parecoxib; (g) cholesterol lowering agents such as HMG-CoA reductaseinhibitors (lovastatin, simvastatin, pravastatin, fluvastatin,atorvastatin, and other statins), sequestrants (cholestyramine andcolestipol), nicotinic acid, fenofibric acid derivatives (gemfibrozil,clofibrat, fenofibrate and benzafibrate), and probucol; (h)anti-diabetic agents such as insulin, sulfonylureas, biguanides(metformin), a-glucosidase inhibitors (acarbose) and glitazones(troglitazone, pioglitazone, englitazone, MCC-555, BRL49653 and thelike); (1) agents that interfere with TNF such as antibodies to TNF(REMICADE®) or soluble TNF receptor (e.g. ENBREL®); (h) anticholinergicagents such as muscarinic antagonists (ipratropium nad tiatropium); (i)antimetabolites such as azathioprine and 6-mercaptopurine, and cytotoxiccancer chemotherapeutic agents.

The entrapped therapeutic agent is, in one embodiment, a cytotoxic drug.The drug can be an anthracycline antibiotic, including but not limitedto doxorubicin, daunorubicin, epirubicin, and idarubicin, includingsalts and analogs thereof. The cytotoxic agent can also be a platinumcompound, such as cisplatin, carboplatin, ormaplatin, oxaliplatin,zeniplatin, enloplatin, lobaplatin, spiroplatin,((−)-(R)-2-aminomethylpyrrolidine (1,1-cyclobutanedicarboxylato)platinum),(SP-4-3(R)-1,1-cyclobutane-dicarboxylato(2-)-(2-methyl-1,4-butanediamine-N,N′)platinum),nedaplatin and(bis-acetato-ammine-dichloro-cyclohexylamine-platinum(IV)). Thecytotoxic agent can also be a topoisomerase 1 inhibitor, including butnot limited to topotecan, irinotecan,(7-(4-methylpiperazino-methylene)-10,11-ethylenedioxy-20(S)-camptothecin),7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin, 9-aminocamptothecinand 9-nitrocamptothecin. The cytotoxic agent can also be a vincaalkaloid such as vincristine, vinblastine, vinleurosine, vinrodisine,vinorelbine, and vindesine. The entrapped therapeutic agent can also bean angiogenesis inhibitor, such as angiostatin, endostatin and TNF.

Nucleic acids are also contemplated for use as the therapeutic agent.DNA and RNA based nucleic acids, including fragments and analogues, canbe used for treatment of various conditions, and coding sequences forspecific genes of interest can be retrieved from DNA sequence databanks,such as GenBank or EMBL. For example, polynucleotides for treatment ofviral, malignant and inflammatory diseases and conditions, such as,cystic fibrosis, adenosine deaminase deficiency and AIDS, have beendescribed. Treatment of cancers by administration of tumor suppressorgenes, such as APC, DPC4, NF-1, NF-2, MTS1, RB, p53, WT1, BRCA1, BRCA2and VHL, are contemplated. Administration of the following nucleic acidsfor treatment of the indicated conditions are also contemplated: HLA-B7,tumors, colorectal carcinoma, melanoma; IL-2, cancers, especially breastcancer, lung cancer, and tumors; IL-4, cancer; TNF, cancer; IGF-1antisense, brain tumors; IFN, neuroblastoma; GM-CSF, renal cellcarcinoma; MDR-1, cancer, especially advanced cancer, breast and ovariancancers; and HSV thymidine kinase, brain tumors, head and neck tumors,mesothelioma, ovarian cancer.

The polynucleotide can be an antisense DNA oligonucleotide composed ofsequences complementary to its target, usually a messenger RNA (mRNA) oran mRNA precursor. The mRNA contains genetic information in thefunctional, or sense, orientation and binding of the antisenseoligonucleotide inactivates the intended mRNA and prevents itstranslation into protein. Such antisense molecules are determined basedon biochemical experiments showing that proteins are translated fromspecific RNAs and once the sequence of the RNA is known, an antisensemolecule that will bind to it through complementary Watson-Crick basepairs can be designed. Such antisense molecules typically containbetween 10-30 base pairs, more preferably between 10-25, and mostpreferably between 15-20. The antisense oligonucleotide can be modifiedfor improved resistance to nuclease hydrolysis, and such analoguesinclude phosphorothioate, methylphosphonate, phosphodiester and p-ethoxyoligonucleotides (WO 97/07784). The entrapped agent can also be aribozyme or catalytic RNA.

Typically, treatment of pathologic conditions is effected byadministering an effective amount or dosage of an anti-alpha-V subunitantibody immunoliposome composition. In some patients and for someconditions, the anti-alpha-V antibody has a therapeutic activity, and inthese situations the amount of antibody administered can range, onaverage, from at least about 0.01 to 500 milligrams of at least oneanti-alpha-V subunit antibody per kilogram of patient per dose, andpreferably from at least about 0.1 to 100 milligrams antibody /kilogramof patient per single or multiple administration, depending upon thespecific activity of contained in the composition. Alternatively, theeffective serum concentration can comprise 0.1-5000 μg/mL serumconcentration per single or multiple administration. Suitable dosagesare known to medical practitioners and will, of course, depend upon theparticular disease state, specific activity of the composition beingadministered, and the particular patient undergoing treatment. In someinstances, to achieve the desired therapeutic amount, it can benecessary to provide for repeated administration, i.e., repeatedindividual administrations of a particular monitored or metered dose,where the individual administrations are repeated until the desireddaily dose or effect is achieved.

For other patients and for other diseases, the anti-alpha-V antibodyserves as a targeting ligand, to direct the liposome and its entrappedtherapeutic drug to a specific site in vivo. In these cases, the dosageof immunoliposome is selected according to the desired serumconcentration of the entrapped therapeutic drug.

For other patients and for other diseases, the anti-alpha-V antibody hasa therapeutic effect and the entrapped drug has a therapeutic effect.The dosage of the immunoliposome composition will then be selectedaccording to the desired serum concentration of the drug and/or theantibody, as can be determined from in vitro cytotoxicity tests and/orin vivo dosing studies.

The dosage administered can vary depending upon known factors, such asthe pharmacodynamic characteristics of the particular agent, and itsmode and route of administration; age, health, and weight of therecipient; nature and extent of symptoms, kind of concurrent treatment,frequency of treatment, and the effect desired. The dosage can be aone-time or a periodic dosage given at a selected interval of hours,days, or weeks.

Any route of administration is suitable, with intravenous and otherparenteral modes being preferred.

In another aspect, the invention contemplates a combined treatmentregimen, where the immunoliposome composition described above isadministered in combination with a second agent. The second agent can beany therapeutic agent, including other drug compounds as well asbiological agents, such as peptides, antibodies, and the like. Thesecond agent can be administered simultaneously with or sequential toadministration of the immunoliposomes, by the same or a different routeof administration.

IV. Examples

The following examples are illustrative in nature and are in no wayintended to be limiting.

Materials

Hydrogenated soy phosphatidylcholine (HSPC) was purchased from LipoidK.G. (Ludwigshafen, Germany). Cholesterol was received from Croda, Inc.(New York, N.Y.) and N-(carbonyl-methoxypolyethylene glycol2000)-1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine, sodium salt(mPEG-DSPE) was received from Genzyme (Cambridge, Mass.). Doxorubicinhydrochloride was received from Meiji Seika Kaisha Ltd. (Tokyo, Japan).

Dithioerythritol (DTE), ethylenediaminetetraacetic acid (EDTA),iodoacetamide (IAC), N-ethylmaleimide (NEM), sodium phosphate monobasic,sodium phosphate dibasic, NaCl, and copper (II) chloride dihydrate werepurchased from Sigma (St. Louis, Mo.). Maleimide-terminated PEG coupledto DSPE (MalPEG-DSPE) was purchased from Avanti Polar Lipids (Alabaster,Ala.). The desalting columns, HiTrap SP HP ion exchange columns, and thesephacryl 300 size-exclusion columns were purchased from AmershamBiosciences (Piscataway, N.J.).

Example 1 Preparation of Liposomes 1. Liposome Preparation

Liposome-entrapped doxorubicin was prepared using methods previouslydescribed (e.g, U.S. Pat. No. 5,013,556). In brief, the lipid components(HSPC, CHOL, mPEG-DSPE at a molar ratio of 56.4:38.3:5.3) weresolubilized in ethanol and added to 250 mM ammonium sulfate solution at60-65° C. The solution was mixed for 1 hour at this elevated temperatureto allow for hydration of the lipid components and formation ofliposomes. The liposomes were downsized below a mean particle size of100 nm by extrusion. The process fluid was diafiltered with ammoniumsulfate solution to remove the ethanol, followed by sucrose solution toremove the ammonium sulfate in the external liposomal phase. A sample ofthe post diafiltration process fluid was submitted for phosphorusconcentration determination and diluted to a target phosphorusconcentration based on the measured value. Doxorubicin was loaded intothe liposomes by incubating the liposomal process fluid with doxorubicindrug solution at 60-65° C. for 1 hour. The resulting drug loadedliposomes were cooled and stored at 2-8° C.

Example 2 Preparation of an Anti-Alpha-V Fab Using a Protease 1. ParentAntibody

The isolated parent antibody, CNTO 95, a heterodimer consisting of SEQID NO: 1 and SEQ ID NO: 2 as disclosed in U.S. Pat. No. 7,163,681; wasdesired as the source of Fab′ used as a targeting-ligand. CNTO95 is afull-length human antibody of the IgG1k type. The monovalent bindingarm, Fab′, to be used represents residues 1-234 or the heavy chain (SEQID NO: 1) and the entire light chain (SEQ ID NO: 2).

2. Preparation of F(ab′)2

Cleavage of CNTO95 with pepsin under conditions to release the Fcportion from the (Fab′)₂ of the antibody was performed. Starting withCNTO95 purified using Protein A chromotography, the antibody wasdiafiltered into 0.1M Citrate pH 4.2 to a final concentration of 10 g/L.Pepsin (Sigma Cat no P6887), reconstituted as a stock solution in thesame buffer, was added at a final concentration of 100U Enzyme/mg IgGand allowed to digest for 90 min at 40° C. The digestion was stopped byraising the pH to 6.0 with Tris-base and the material was filter using a0.22 um cut-off membrane.

CNTO95 F(ab′)2 proved to have some affinity for protein A column andtherefore, to improve the yield, the pepsin digest was first purifiedusing cation exchange chromatography Sepharose HP (GE Healthcare,Piscataway N.J., Cat. No. 17-1087-01) prior to it being passed overProtein A conjugated beads (MABSELECT™, GE Healthcare, Piscataway N.J.,Cat. No. 17-5199-01) in a flowthrough mode.

The protein was further purified by anion exchange using a Q Sepharose™XL (GE Healthcare, Cat No. 17-5072-01) in a flowthrough mode. Theproduct is final purified by ultrafiltration using a 30 kDa MW cut-offmembrane and finally concentrated to 10 mg/mL with 30 mM Na₂HPO₄ pH 6.0.

3. Reduction of F(ab′)₂

F(ab′)₂ was diluted with saline to a target protein concentration of 3.5mg/mL. The pH of the protein solution was adjusted to 6.5 using 1Msodium phosphate monobasic and 1M sodium phosphate dibasic. A 150 mMdithioerythritol (DTE) stock solution was prepared by dissolving the DTEin the correct volume of water. The volume of 150 mM DTE solution toachieve a 13 mM concentration when added to the protein solution wascalculated. The protein was placed in a water bath set to 40° C.Sufficient time was allowed for the protein solution to reach 40° C.prior to adding the reducing agent. The correct volume of DTE was addedto the protein solution and incubated at 40° C. for 60 minutes whilemixing. At the end of the incubation time the protein solution wasplaced on ice.

DTE was removed by passing the protein solution over a desalting column.The column was prepacked with Sephadex G-25 with a diameter and heightof 2.6 and 10 cm respectively. Up to 20 ml of solution could be loadedon the column for separation of protein from reducing agent. For volumesgreater than 20 mL, the desalting step was done in batches. The runningbuffer used was 30 mM sodium phosphate buffer, pH 6.0 that was argonsparged. The low salt concentration of the running buffer allowed forefficient binding of the protein to the ion exchange column in the nextstep. As a note, ultra pure water (Milli Q system) was used in makingall solutions and buffers to minimize any potential contamination ofheavy metals that could affect the reoxidation rate. The flow rate overthe column was 10 mL/min.

4. Ion Exchange Step

The protein solution was next loaded onto a HiTrap SP HP ion exchangecolumn. The column size was based on loading approximately 10 mg ofprotein per 1 mL of column packing. The flow rate during the loadingstep was ½ a column volume per minute. After loading all of the protein,the column was washed with 10 column volumes of 30 mM sodium phosphatebuffer, pH 6.0 that was argon sparged in order to remove any residualDTE. Next, the column was washed with 10 column volumes of 30 mM sodiumphosphate buffer, pH 6.0 that was air sparged. The protein was elutedfrom the column with 30 mM sodium phosphate buffer, 60 mM NaCl, pH 6.0that was air sparged. The purpose for sparging the buffers in air atroom temperature was to saturate the buffers with oxygen and make theprocess reproducible.

The pH of the eluted protein solution was checked and adjusted ifnecessary to 6.0. The protein concentration of the protein wasdetermined and diluted to a value of 1.02 mg/mL with the same bufferused to elute the protein (30 mM sodium phosphate buffer, 60 mM NaCl, pH6.0 that was air sparged). The protein solution was placed in a glasscontainer with the appropriate capacity to minimize the headspace in thecontainer and placed in a water bath set to 20° C.

5. Reoxidation Process

A 63.75 μM CuCl₂ stock solution was prepared. This low concentration wasachieved by first making a 15.94 mM stock solution by dissolving theappropriate amount of CuCl₂ in water and performing successive dilutionsin water until the final concentration was achieved. 20 μL of the 63.75μM CuCl₂ stock solution was added for every 1 mL of protein solution.After mixing, the protein concentration was 1.00 mg/mL and the CuCl₂concentration was 1.25 μM.

Samples were taken throughout the reoxidation process to monitor theextent of the reoxidation. The samples were run on an HPLC system with asize exclusion column and a running buffer containing SDS. The Fab′ peakwas resolved from the heavy and light chain peaks allowing for thequantitation of the % Fab′ at the time the sample was taken. Based onthese results, the time for reoxidation was determined. The time coursefor the reoxidation process was nearly identical for all batches madewith an optimal time for reoxidation of 320 minutes.

6. Maleimide-Terminated PEG Conjugated to DSPE Insertion into Pre-FormedLiposomes

MalPEG-DSPE was dissolved in water for injection at a concentration of10 mg/mL. The volume of MalPEG-DSPE solution to add to the liposomalsolution was calculated based on 1) the phosphorus concentration of thepost drug loaded liposomes, 2) the assumption that each liposome iscomprised of 80,000 phospholipids and 3) 800 MalPEG-DSPE molecules areinserted per liposome. The calculated amount of MalPEG-DSPE solution wasthen added to the appropriate amount of post drug loaded liposomalsolution, prepared as in Example 1, and incubated at 60 to 65° C. for 1hour followed by cooling in an ice bath. 9% NaCl solution was added tothe process fluid at a volume ratio of 1 to 9 to bring the solution upto 0.9% NaCl concentration. Addition of salt was deemed necessary tominimize any potential Fab′ denaturation under low salt conditionsduring the conjugation step. The solution pH was adjusted to 6.0 usingeither 1M sodium phosphate monobasic or 1M sodium phosphate dibasic. Thepreparation of the inserted liposomal material was typically completed acouple of hours prior to the conjugation step to minimize any potentialinactivation of MalPEG-DSPE over time.

7. Conjugation Step

At the end of reoxidation, the appropriate volume of protein solutionwas added to the post MalPEG-DSPE inserted liposomes to begin theconjugation process. The amount of protein required was calculated basedon 1) the desired Fab to liposome ratio, 2) the assumption that eachliposome is comprised of 80000 phospholipid molecules, 3) phosphorusconcentration of the post inserted solution and 4) the assumption that50% of the protein in solution will conjugate as Fab. The lastassumption was based on small-scale optimization work. For some of thelater batches produced, EDTA solution was added to the liposomal andprotein solutions to achieve a 1 mM concentration in the final mixture.This addition minimized any potential reoxidation during the conjugationprocess. Conjugation was at room temperature for 2 hours followed byovernight storage at 2-8° C.

8. Quenching and Final Column Purification

The conjugated liposomal formulations were quenched at a 1 mM cysteineconcentration for 10 minutes prior to loading on the size exclusioncolumn. The column contained sephacryl-300 packing with adiameter/height of 1.6/60 or 2.6/60 cm depending on the volume ofsolution to load on the column. A large volume (20% of the columnvolume) could be loaded on the column due to the large size differencebetween the liposomes and the unconjugated protein. The column removedunconjugated protein, unreacted cysteine and unencapsulated doxorubicin.The running buffer was 10 mM histine in saline, pH 6.5. The liposomalfraction was concentrated to a target drug concentration of 2.0 mg/mLwith a centri prep concentrator with a 100K MWCO membrane at 2800 rpm.

The final formulations were submitted for potency, % drug encapsulation,particle size, pH, % Fab insertion and endotoxin. The reoxidationprocess was evaluated through analysis of both blocked and conjugatedsamples taken throughout the process. The samples were analyzed by SDSgel electrophoresis and the bands were quantified by densitometrymeasurements. Tables 1A-1B below summarize the characteristics of twobatches of liposomes.

TABLE 1A Characteristics of targeted liposomes in Batch 1 Batch 1:Targeting Particle ligand:liposome Potency % drug Size % ratio (mg/mL)encapsulation (nm) insertion 15:1 2.21 99 85 98.7 40:1 2.19 99 87 98.890:1 2.09 97 88 99.7

TABLE 1B Characteristics of targeted liposomes in Batch 2 Batch 2:Targeting Particle ligand:liposome Potency % drug Size % ratio (mg/mL)encapsulation (nm) insertion 15:1 2.15 99 85 98.4 40:1 2.12 98 87 98.590:1 2.24 98 90 99.5

Example 3

In Vitro Plasma Dissolution of Targeting Ligand from Liposomes

The purpose of this study was to evaluate in-vitro plasma stability ofalpha-integrin targeted liposomes with prepared by the method of Example1 using a Fab as targeting ligand at a ratio of 15:1 ligand to liposome,at 37° C.

1. Preparation of I-125 Fab-PEG-DSPE

Fab-PEG-DSPE was mixed with prepared iodobeads for 20 minutes and thenplaced over 2 desalting columns to separate the I-125 Fab-PEG-DSPE fromfree I-125. The concentration of the protein was determined by UVabsorbance at 280 nm for each of the protein fractions collected. Theprotein fractions were pooled from each column with the highest proteinconcentration

2. Preparation of 125-I-Fab′ Conjugated STEALTH® Liposomal Doxorubicin

Liposomes with entrapped doxorubicin were prepared as set forth inExample 1 and then incubated with sufficient I-125Fab-PEG-DSPE togenerate a 15:1 Fab/liposome ratio at 60° C. for 1 hour to allowinsertion of the I-125Fab-PEG-DSPE conjugate. At the end of theincubation the solution was cooled and subsequently stored at 2-8° C.Post-insertion material was passed through sepharose-CL-4B column toremove un-inserted Fab-PEG-DSPE. The final formulation was characterizedfor size, pH, doxorubicin concentration, doxorubicin encapsulation,Fab-PEG-DSPE insertion and Fab-PEG-DSPE concentration. The liposomecharacteristics are summarized in Table 2A.

TABLE 2A Fab-PEG-DSPE Fab:Liposome Potency Particle Size % Drug %Fab-PEG- Concentration Ratio (mg/mL) (nm) 90°/30° Encapsulation DSPEInsertion (μg/mL) 15:1 2.02 134/153 99 97 74.05

3. Preparation of Sepharose CL-4B Columns

Two 28×1.2 cm columns containing sepharose CL-4B were prepared, eachwith a bed volume of 32 mL. The columns were pre-conditioned with 1 mLplacebo liposomes and 1 mL rat plasma to block any potential bindingsites on the column and therefore minimize any binding of samples to thecolumn. The elution buffer used for all samples was saline containing0.02% sodium azide. The columns were evaluated to assess whetherliposomes could be separated from plasma proteins. Placebo liposomeswere combined with rat plasma and loaded on the sepharose CL-4B columns.Eluted fractions were collected from each column and the presence ofliposomes or proteins was detected by absorbance at 280 nm. Columnprofiles showed good separation between liposomes and plasma. Therecovery of sample from the column was determined by comparing theactivity of the unseparated sample to the total activity of all of thefractions collected for that sample. The recovery was greater than 90%for all samples.

4. In Vitro Fab-PEG-DSPE Dissociation in Human Plasma

Human plasma was mixed with 125-I-labeled, targeted liposomes andincubated at 37° C. over 96 hours. At given time points (0, 1, 4, 8, 24,48, 72, 96 hours) a sample was removed and loaded onto sepharose CL-4Bcolumn. Fractions (1 mL each) were collected from the column and thetotal radioactivity for each fraction was counted using a gamma counterfor 125-I radioactivity.

5. Results

The elution profiles of the 125-I Fab-PEG-DSPE conjugate for all sampletime points were similar (not shown), with minor profile differences dueto column packing efficiencies. Radioactivity in the liposomal fractionwere recovered within a very narrow range within 2-3 fractions andtypically within 6-9 mL of total eluent collected. Plasma fractionseluted from the sepharose CL-4B column over at least 12 fractions, dueto the wide size range of plasma proteins. Over all time points, theliposomal and plasma fraction were distinguishable from each other. Inorder to calculate percentage of dissociated Fab-PEG-DSPE, theradioactivity from the liposomal (or plasma) fractions were combined toprovide a total amount of Fab-PEG-DSPE. That total was assessed as aratio of total radioactivity in the sample applied to the SepharoseCL-4B column. FIG. 1 and Table 3 show the percentage of Fab-PEG-DSPEconjugate remaining in the liposomes and the percentage of conjugatedissociated from the liposomes as a function of incubation time.

TABLE 3 Percent of 125-I Fab- PEG-DSPE remaining the liposome afterincubation in human plasma at 37° C. % of Fab-PEG- % of Fab-PEG- DSPEConjugate in DSPE Conjugate in Time (hr) Liposomal Fraction PlasmaFraction 0 90 10 1 89 11 4 89 11 8 87 13 24 89 11 48 88 12 72 88 12 9688 12

The data from this study indicated that αv-targeted liposomes are stablein human plasma over 96 hours incubation at 37° C.

Example 4 In Vitro Binding and Internalization

This study evaluates the ability of integrin-targeted liposomes toachieve ligand mediated specific binding, internalization, and cellcytotoxicity in tumor cells bearing a humanized αVβ3/5 integrinreceptor, as compared to liposomes lacking a targeting ligand.

1. Cells and Cell Media

Several human tumor cells bearing human αVβ3/5 integrins were used: (1)A375.S2, human melanoma cell line; (2) MDA-MB-231, Human BreastCarcinoma Cell line; (3) A2780, Human Ovarian Carcinoma Cell line; (4)HT29, Human Colon Carcinoma Cell line; (5) A549, Human Lung CarcinomaCell line. As CNTO95 does not bind murine αVβ3/5 integrin, a murinemelanoma cell line, B16F10, was used as a negative control in the study.

The media for each cell line was as follows:

-   -   1. A375.S2 cell, MEM (Minimum Essential Medium, ATCC Cat No.        30-2006) with addition of 10% Fetal Bovine Serum (FBS, ATCC, Cat        No. 30-2021).    -   2. MDA-MB-231 cell, Leibovitz's L-15 Medium, (ATCC Cat No.        30-2008) with addition of 10% FBS.    -   3. A2780 cell, RPMI Medium 1640 (Gibco, Cat No. 22400-089HT29)        with addition of 10% FBS.    -   4. A549 cell, F-12K Medium (ATCC, Cat No. 30-2004) with addition        of 10% FBS.    -   5. B16-F10 cell, DMEM (Dulbecco's Modified Eagles's Medium, ATCC        Cat No. 30-2002), with addition of 10% FBS.

Cell viability was assayed using the CellTiter 96 AQueous One SolutionCell Proliferation Assay from Promega (Cat No. G3581). A Spectra Max 250plate reader was used, with a reading wavelength of 490 nm. ConfocalMicroscopy was done using a Nikon, Eelipse, E600. An Eppendorfcentrifuge 5804 was used.

2. Liposome Compositions

Liposomes were prepared as described in Example 1, except in twoaspects. First, Dextran Alexa Fluor 488 (Cat No. D-22910, from MolecularProbes) was included in the hydration buffer during the passiveencapsulation step of liposome formation, and after the sizing stepdialysis was used to remove any unencapsulated Alexa. Second, instead ofinserting Fab-PEG-lipid into the liposome bilayer, maleimide-PEG-lipid(MalPEG-DSPE) was inserted into the liposome bilayer at approximately800 MalPeg-DSPE per liposome at 60° C. for 1 Hr. The insertion step wasfollowed by the conjugation of the Fab to the reactive end of thelipopolymer. The appropriate amount of Fab was added to the MalPEG-DSPEinserted liposomes to achieve a 90:1 anti-alpha-V-Fab targeting ligandsper liposome ratio. These liposomes were used in the binding andinternalization studies described below.

Liposomes bearing 15:1, 40:1, 90:1 and 180:1 alpha-integrin Fabtargeting ligands per liposome and containing doxorubicin were preparedas described in Example 1 and 2. These targeted-liposomes were used inthe cytotoxicity assay using the various cells lines, as describedbelow.

3. Binding and Internalization Studies

A375.S2 cells were harvested by scraping and then resuspended to obtainindividualized cells and rejuvenated for 1 hour, at 37° C. About 1million cells of each tumor type were counted and distributed intoindividual centrifugation tubes. The tubes were spun to obtain a cellpellet.

For binding only studies, the cells were cooled to 4° C. by immersingthe cell tubes in ice for 10 minutes and then treating with the targetedliposome composition containing a fluorescent marker (Dextran AlexaFluor 488) at 4° C. for 30 minutes, with mild shaking (140 rpm). Afterthe 30 minute incubation period, 1 mL of cold serum free media wasadded, the mixture was vortexed briefly, and the centrifuged. The cellpellet was resuspended with cold serum free media, shaken vigorously(440 rpm) at 4° C. for 10 minutes, and then centrifuged to recover thecell pellet. The cell pellet was left in about 100 μL of cold media andabout 8 μL was taken for observation under a confocal microscope. Allsteps, except observation under the confocal microscope, were conductedat 4° C.

For binding and internalization, the cells were treated with thetargeted liposome formulation at 37° C. for 10 min with mild shaking(140 rpm). Cells were treated with the liposome formulations containingeither a doxorubicin payload or a fluorescent marker (Dextran AlexaFluor 488). Treatment was terminated by adding 1 mL of washing media(serum free), vortexing briefly, and centrifuging to recover a cellpellet The cells in the pellet were resuspend in washing media,vigorously shaken for 10 minutes at 37° C. and then centrifuged again(440 rpm). The cell pellet was left in 100 μL of media, an aliquot of 8μL was taken and placed on a glass slide for observation under aconfocal microscope.

Binding and internalization of the targeted liposomes to A375.S2 cellswas evaluated after various incubation times of the cells and theliposome formulation. Results are shown in FIGS. 2-5.

In FIGS. 2A and 2C, confocal microscopy results show that the targetedliposome formulation containing a fluorescent marker (Dextran AlexaFluor 488) binds specifically to A375.S2 cells at 4° C. in vitro whilethe corresponding untargeted liposome formulation containing fluorescentmarker does not. FIGS. 2B and 2D show images of the cells in“differential interference contrast” mode (DIC) and provides a referenceon cell locations for FIGS. 2A and 2C. All subsequent Figures have a DICpictures that correspond to the confocal image for reference. Confocalmicroscopy results shown in FIGS. 3A through 3H that liposomes bearing90:1 alpha-integrin Fab targeting ligands per liposome as described inExamples 1 and 2 (“targeted liposome formulations”) specifically bind toA375.S2 cells and internalize into the same cells in vitro. FIGS. 3A and3B show cells that were not treated with drug and, as expected, noevidence of binding or internalization was observed. When the cells weretreated with free doxorubicin (i.e., nonliposomal drug) in FIGS. 3C and3D, drug internalization is evident, however, the diffuse fluorescencepattern suggests the mechanism of drug internalization was nonspecificdiffusion of drug across the cell membrane. FIGS. 3E and 3F show cellstreated with untargeted liposomes containing doxorubicin. No evidence ofbinding or internalization of these liposomes was observed. Finally,specific binding and internalization of was observed for the targetedliposome formulation (see FIGS. 3G and 3H) and the fluorescence patternis marked by regions of high fluorescence intensities on the surface andinside the cytoplasm indicative of liposome internalization under thetreatment regime described above.

FIG. 4A through 4J show a timecourse study following internalization ofthe Dextran Alexa Fluor 488 fluorescent marker and doxorubicin (24 hourtimepoint only). As the time post-treatment increases, evidence ofinternalization and penetration into the cytoplasm becomes more clear.More importantly, this data suggests the presence of the fluorescentmarker in the cytoplasm may be due to liposome internalization and notfluorescent marker leakage from liposomes followed by diffusion sincethe fluorescent marker used in this study cannot diffuse across the cellmembrane.

FIGS. 5A through 5H show results from a similar experiment shown inFIGS. 3A through 3H. The one change in the experimental conditions wasthe use of a murine cell line B16.F10 that does not express alpha-Vintegrins on its cell surface. The purpose of this experiment was toshow that liposomes bearing 90:1 alpha-integrin Fab targeting ligandsper liposome as described in Examples 1 and 2 only bind to cellsexpressing alpha-V. The confocal microscopy images demonstrate that thisis the case. No binding was observed in this cell line which suggestsalpha-V targeted liposomes have a high degree of specificity for alpha-Vover-expressing tumor types.

4. Method of Cytotoxicity Assay

Cells were harvested by scraping and then resuspended at 37° C. for 1hour to obtain individualized cells. About 1 million cells of each tumortype were counted and placed in individual centrifugation tubes. Thetubes were centrifuged to obtain a cell pellet. The cells were thenincubated with the targeted liposome compositions containing doxorubicinfor 10 minutes at 37° C., with mild shaking, 140 rpm. Cells were treatedwith a quantity of liposomes sufficient to give a doxorubicinconcentration of 40 μg/mL. After the minute period, 1 mL of washingmedia (serum free) was added, the cells were vortexed briefly and thencentrifuged to obtain a cell pellet. The pellet was resuspended in serumfree washing media, vigorously shaking for 10 minutes at 37° C., 440rpm. After centrifuging again, 1 mL of media containing 10% fetal bovineserum was added. Cells from each tube were seeded on a plate at aconcentration of 2000 cell/well, in triplicate for each point. The platewas incubated for 3 and 6 days and then a cell viability assay for cellgrowth inhibition was conducted.

The images (FIGS. 6-9) from the binding study show that alpha-V targetedliposomes specifically bind, and are internalized by the αVβ3/5 integrinpositive A375.S2 human melanoma cells. Internalization was timedependent and at longer exposure times, cells internalized a greaternumber of liposomes. Internalization occurs rapidly, with cells exposedto targeted liposomes for 10 minutes achieving internalization ofliposomes into the cell cytoplasm. The presence of liposomes in thenucleus of the cells was also observed in the confocal microscopyimages.

The alpha-integrin targeted liposomes displayed specific cytotoxicitytoward human αVβ3/5 integrin positive cell lines, including A375.S2,MDA-MB-231, and A2780. As expected, the alpha-V targeted liposomes hadno binding to the murine cell line used as a negative control, B16.F10cell.

The data obtained from the cytotoxicity studies was used to determinemolar concentration of each doxorubicin-containing liposomes formulationthat produced 50% of the maximum possible response (IC₅₀). IC₅₀ valueswere determined for doxorubicin in free form, doxorubicin entrapped inliposomes lacking the targeting antibody fragment, and doxorubicinentrapped in liposomes bearing targeting ligands at densities of 40:1and 90:1 when applied to melanoma tumor cells (A375.S3), breast cancercells (MDA-MD-231), human ovarian cancer cells (A2780), colon cancercells (HT29), lung cancer cells (A549), and the non-integrin bearingB16-F10 cells. The values are summarized in Table 4 where the “Increase”corresponds to ratio of IC₅₀ value for liposome-entrapped doxorubicin tothe IC₅₀ value for integrin-targeted liposome-entrapped doxorubicinbearing 90:1 ligand:liposome.

TABLE 4 IC₅₀ Values of doxorubicin in various formulations integrintargeted integrin targeted liposome- liposome- Tumor Cell liposome-entrapped dox entrapped dox Line free dox entrapped dox 40:1 90:1Increase melanoma >200 32 15 7 29 AS375.S2 breast cancer >200 110 45 525 MDA-MS-231 ovarian >200 9 12 7 29 cancer A2780 colon cancer >200 >200180 >200 1.1 HT29 Lung cancer >200 >200 >200 >200 1 A549 Murine >200 33na >200 1 melanoma B16-F10

These data show that certain human tumor derived cell lines aresensitized to the alpha-V-targeted liposomes.

Example 5 Pharmacokinetic Profiles of Integrin-Targeted Liposomes inMice

The objective of this study was to determine the pharmacokineticprofiles of liposomes bearing Fab′ targeting ligands, prepared as inExample 1 and 2, for α_(v) integrin after a single intravenous (IV)bolus administration in female CD-1 mice. Four different formulationswith varying Fab′ to liposome ratios, i.e., 15, 40, 90, and 180 weretested and compared to the pharmacokinetic profile of a liposomeslacking a targeting ligand.

1. Liposome Compositions

Liposomes lacking an integrin targeting ligand, referred to as “S-DOX”,were prepared as described in Example 1.

Alpha-V-targeted liposomes, referred to as “Fab′ S-DOX”, were alsoprepared as described in Example 2. The four integrin-targeted liposomeformulations were:

-   -   15:1 Fab′ to liposome ratio—The doxorubicin concentration was        2.15 mg/mL and encapsulation was 99%. The average diameter of        liposomes in the final formulation was 85 nm.    -   40:1 Fab′ to liposome ratio—The doxorubicin concentration was        2.12 mg/mL and encapsulation was 98%. The average diameter of        liposomes in the final formulation was 87 nm.    -   90:1 Fab′ to liposome ratio—The doxorubicin concentration was        2.24 mg/mL and encapsulation was 98%. The average diameter of        liposomes in the final formulation was 90 nm.    -   180:1 Fab′ to liposome ratio—The doxorubicin concentration was        2.03 mg/mL and encapsulation was 95%. The average diameter of        liposomes in the final formulation was 90 nm.        The test formulations are summarized in the Table 5.

TABLE 5 Summary of Liposome Test Formulations Fab′ to Drug Dosing GroupNo. of Dose liposome Dose Concentration No. Animals Route Formulationratio (mg/kg) (mg/mL) 1 18 IV S-DOX 0 2 0.2 2 18 IV Fab′-S-DOX 15:1 20.2 3 18 IV Fab′-S-DOX 40:1 2 0.2 4 18 IV Fab′-S-DOX 90:1 2 0.2 5 18 IVFab′-S-DOX 180:1  2 0.2Sterile saline obtained from a commercial source (Abbott Labs, Lot31-115-JT, expiration date January 2007) was used for drug dilutionprior to administration.

2. Study Design

Sixty female CD-1 mice (Charles River Laboratories, Hollister, Calif.),approximately 20 to 26 g body weight were used for the study . Animalswere maintained in isolator cages on a 12-hour light-and-dark cycle.Food and water were available ad libitum.

All animals were administered a single bolus injection of one of thetest formulations via a lateral tail vein. Dose volumes were calculatedfor each individual animal by body weight, ranging from 0.21 to 0.26 mL.Mice were warmed prior to injection in a rodent hotbox. Doxorubicin dosefor all treatment groups was 2 mg/kg.

Clinical observations and body weights were recorded prior to dosing.Animals were observed daily thereafter for morbidity and mortality.

Blood samples (about 0.6 mL each) were collected from three mice pertime point (5 min, 4, 8, 24, 48, and 96 hr). Blood samples werecollected either via cardiac puncture or the hepatic portal vein underinhaled anesthesia (oxygen/Isoflurane) into heparin-coated syringes andimmediately transferred into a polypropylene eppendorf tube. The bloodsample collection procedure was terminal. Blood samples were then storedon wet ice until centrifugation at 10,000 RPM for 5 minutes at ˜4° C.Plasma samples were collected and stored at −20° C. Total doxorubicinconcentrations were analyzed by LC/MS.

Since each plasma sample was obtained from an individual animal, thepharmacokinetic (PK) parameters were calculated using the mean plasmadoxorubicin concentrations and therefore no standard deviations arepresent for the PK parameters. All PK parameters were calculated usingWINNONLIN version 4.1 (Pharsight Corp., Mountain View, Calif.).

3. Results

Pharmacokinetic profiles of all test formulations are shown in FIG. 10and are summarized in Table 6.

TABLE 6 Plasma PK Parameters of Doxorubicin Following a Single IVAdministration of Fab′-S-DOX Formulations C_(max) AUC_(last) T_(1/2) ClFormulation (±SE, ng/mL) T_(max) (ng-h/mL) (h) (mL/h) S-DOX 37,133 ±1,868 5 min 554,035 13.6 0.0838 Fab′-S-DOX 15:1 30,133 ± 1,009 5 min562,945 14.4 0.0883 Fab′-S-DOX 40:1 32,467 ± 1,225 5 min 536,216 11.60.0928 Fab′-S-DOX 90:1 28,833 ± 448   5 min 440,048 15.5 0.1005Fab′-S-DOX 180:1 8,993 ± 499  4 h  186,095 14.3 0.2480

Results showed that plasma concentration peaked by the first samplingtime point (within 5 min) for all Fab′-S-DOX formulations except for theone with 180 Fab′ per liposome, which peaked at the 4 h time point.C_(max) was 37133, 30133, 32467, 28833, and 8993 ng/mL for Fab′-S-DOXformulations containing Fab′/liposome ratios of 0, 15, 40, 90, and 180,respectively. The AUC_(last) values were similar for those withFab′/liposome ratios of 0, 15, and 40 (536216 to 562945 ng-h/mL),slightly lower for the ratio of 90 (440048 ng-h/mL), and considerablylower for the ratio of 180 (186095 ng-h/mL). Plasma half-lives were alsosimilar for 4/5 test formulations, ranging between 13.6 to 15.5 h, andthe formulation with the Fab′/liposome ratio of 40 had a t_(1/2) of 11.6h. Drug clearance was also the greatest for the 180 Fab′ formulation(0.2480 mL/h) followed by the 0 to 90 Fab′ formulations (0.0838 to0.1005 mL/h).

In this mouse study, similar plasma PK profiles/parameters were observedfor the Fab′-S-DOX formulation with Fab′/liposome ratio of 15 or 40 whencompared to the non-targeted S-DOX. Fab′-S-DOX formulation withFab′/liposome ratio of 90 had slightly lower C_(max) and AUC value.Formulation with 180 Fab′/liposome ratio had the lowest (about 70%lower) C_(max) and AUC.

Example 6 Pharmacokinetic Profiles of Integrin-Targeted Liposomes inRats

The objective of this study was to compare the plasma pharmacokinetic(PK) profile of various Fab′ STEALTH liposomal doxorubicin formulations(Fab′-S-DOX) using surface-conjugated Fab′ as a targeting ligand forα_(v) integrin in rats. The Fab′ to liposome ratio in the liposomes were15:1, 30:1, 60:1, and 90:1.

1. Liposome Compositions

Liposomes lacking an integrin targeting ligand, referred to as “S-DOX”,were prepared as described in Example 1.

Integrin-targeted liposomes, referred to as “Fab′ S-DOX”, were alsoprepared as described in Example 1 and 2. The four integrin-targetedliposome formulations were:

-   -   15:1 Fab′ to liposome ratio: the doxorubicin concentration was        2.23 mg/mL and encapsulation was 99%. The average diameter of        liposomes in the final formulation was 84 nm.    -   30:1 Fab′ to liposome ratio: the doxorubicin concentration was        2.26 mg/mL and encapsulation was 99%. The average diameter of        liposomes in the final formulation was 86 nm.    -   60:1 Fab′ to liposome ratio: the doxorubicin concentration was        2.28 mg/mL and encapsulation was 98%. The average diameter of        liposomes in the final formulation was 88 nm.    -   90:1 Fab′ to liposome ratio: the doxorubicin concentration was        2.22 mg/mL and encapsulation was 97%. The average diameter of        liposomes in the final formulation was 89 nm.        The test formulations are summarized in the Table 7.

TABLE 7 Summary of Liposome Test Formulations Fab′ to Drug Dosing GroupNo. of Dose liposome Dose Concentration No. Rat Route Formulation ratio(mg/kg) (mg/mL) 1 4 IV S-DOX 0 1 0.4 2 4 IV Fab′-S-DOX 15:1 1 0.4 3 4 IVFab′-S-DOX 30:1 1 0.4 4 4 IV Fab′-S-DOX 60:1 1 0.4 5 4 IV Fab′-S-DOX90:1 1 0.4Sterile saline obtained from a commercial source (Abbott Labs, LotC665851, expiration date May 2007) was used for drug dilution prior toadministration.

2. Study Design

Twenty male CD rats (Charles River Laboratories, Indianapolis, Ind.)were used for the study. The average body weight was 273 g at dosing.Animals were maintained in cages on a 12-hour light-and-dark cycle. Foodand water were available ad libitum.

All animals were administered a single bolus intravenous (IV) injectionof the test formulation via a lateral tail vein. Dose volumes werecalculated for each individual animal by body weight, which ranged from0.65 to 0.72 mL. Rats were warmed prior to injection in a rodent hotbox.The doxorubicin dose for all treatment groups was standardized to 1mg/kg. Clinical observations and body weights were recorded prior todosing. Animals were observed daily thereafter for morbidity andmortality.

Blood samples (˜0.6 mL each) were collected from four rats per timepoint (2-5 min, 4, 8, 24, 48, and 72 hours). Blood samples werecollected under inhaled anesthesia (oxygen/Isoflurane) via the tail veinor orbital sinus into heparin-coated syringes and immediatelytransferred into a 1 mL polypropylene collection tube. Blood sampleswere then stored on wet ice until centrifugation at approximately 10,000RPM for 5 minutes at ˜4° C. Plasma samples were collected and stored at−20° C. and total doxorubicin concentration was analyzed by LC/MS.

All PK parameters were calculated using WINNONLIN version 4.1 (PharsightCorp., Mountain View, Calif.) and a non-compartment model. Comparison ofPK parameters, i.e., AUC_(last), t_(1/2), and observed clearance amongtest formulations was performed by one-way ANOVA Tukey analysis.

3. Results

Pharmacokinetic profiles of all test formulations are shown in FIG. 11and the PK parameters are summarized in Table 8.

TABLE 8 Plasma PK Parameters of Doxorubicin Following a Single IVAdministration of Fab′-S-DOX Formulations (Mean ± SD) Conc. at 5 minAUC_(last) T_(1/2) Cl Formulation (ng/mL) (μg-h/mL) (h) (mL/h) S-DOX27,650 ± 3,240  673 ± 76.6 28.3 ± 1.3 0.3426 ± 0.04 Fab′-S-DOX 15:130,075 ± 2,791 370 ± 114 24.4 ± 3.8 0.6964 ± 0.20 Fab′-S-DOX 30:1 27,225± 3,646 486 ± 129 26.1 ± 3.0 0.5119 ± 0.13 Fab′-S-DOX 60:1 21,450 ±1,984  176 ± 51.5 12.7 ± 3.0 1.6359 ± 0.51 Fab′-S-DOX 90:1 25,425 ±3,538 91.6 ± 8.38  4.58 ± 0.45 2.9527 ± 0.29

Results showed plasma concentration peaked at the first sampling timepoint (within 5 min) for all targeted and non-targeted S-DOXformulations. C_(max) was also similar for 4/5 formulations, whichranged from 25,425 to 30,075 ng/mL but slightly lower for the Fab′-S-DOX60:1 (21,450 ng/mL). At the 24-h time point, the plasma concentrationwas the greatest for S-DOX (9,953 ng/mL) followed by Fab′-S-DOX 30:1(7,063 ng/mL), Fab′-S-DOX 15:1 (4,600 ng/mL), Fab′-S-DOX 60:1 (1,317ng/mL) and then Fab′-S-DOX 90:1 (222 ng/mL). Similar trend continued forup to 72 h, and drug concentration was un-detectable for Fab′-S-DOX 90:1starting at the 48 h time point. The AUC_(last) value was also thegreatest for S-DOX (673 μg-h/mL) followed by Fab′-S-DOX 30:1 (486μg-h/mL), Fab′-S-DOX 15:1 (370 μg-h/mL), Fab′-S-DOX 60:1 (134 μg-h/mL)and then Fab′-S-DOX 90:1 (91.6 μg-h/mL). The corresponding t_(1/2) was28.3, 26.1, 24.4, 12.7, and 4.58 h, and the corresponding clearance was0.3426, 0.6964, 0.5119, 1.6359, and 2.9527 mL/h.

PK profiles of Fab′-S-DOX formulation vs. S-DOX was performed using theone-way ANOVA Tukey analysis. For AUC_(last), S-DOX was not differentfrom Fab′-S-DOX 30:1 but was significantly greater than Fab′-S-DOX 15:1(p<0.01), Fab′-S-DOX 60:1 (p<0.001), and Fab′-S-DOX 90:1 (p<0.001). Fort_(1/2), there was no difference between S-DOX and Fab′-S-DOX 15:1 orFab′-S-DOX 30:1 but t_(1/2) was significantly longer for S-DOX thanFab′-S-DOX 60:1 (p<0.001) and Fab′-S-DOX 90:1 (p<0.001). Similarly, theclearance was also significantly greater for the Fab′-S-DOX 60:1 andFab′-S-DOX 90:1 vs. S-DOX (p<0.001).

In this rat PK study, it was demonstrated that Fab′-S-DOX formulationwith Fab′ to liposome ratio of 30:1 had a PK profile similar to thenon-targeted liposomal doxorubicin formulation. Fab′-S-DOX formulationwith Fab′ to liposome ratio of 15 was also similar to that of theliposome formulation having a ratio of 30:1 targeting ligands/liposome.Formulations with Fab′ to liposome ratio of 60 or 90 had significantlylower AUC, shorter half-life and greater clearance when compared to thenon-targeted formulation.

Example 7 Antitumor Activity of αV-Integrin-Targeted Liposomes in HumanMammary Carcinoma Xenografts

The purpose of this study was to evaluate the antitumor activity ofα_(v)-integrin targeted Fab′ conjugated liposomal doxorubicin(Fab′-S-DOX) on MDA-MB-231 human mammary carcinoma xenografts. Threeformulations of Fab′-S-DOX with Fab′ to liposome ratio of 15, 40, and 90were used in the study.

1. Liposome Compositions

Liposomes lacking an integrin targeting ligand, referred to as “S-DOX”,were prepared as described in Example 1.

Alpha-V-targeted liposomes, referred to as “Fab′ S-DOX”, were alsoprepared as described in Example 1 and 2. The three integrin-targetedliposome formulations were:

-   -   15:1 Fab′ to liposome ratio. The doxorubicin concentration was        2.15 mg/mL and encapsulation was 99%. The average diameter of        liposomes in the final formulation was 85 nm.    -   40:1 Fab′ to liposome ratio. The doxorubicin concentration was        2.12 mg/mL and encapsulation was 98%. The average diameter of        liposomes in the final formulation was 87 nm.    -   90:1 Fab′ to liposome ratio. The doxorubicin concentration was        2.24 mg/mL and encapsulation was 98%. The average diameter of        liposomes in the final formulation was 90 nm.

2. Xenograft Preparation

Female athymic nu/nu homozygous mice (Harlan Laboratories, Indianapolis,Ind.), approximately 5-6 weeks old, were used for the study. The meanbody weights were approximately 22 grams. Animals were maintained inisolator cages on a 12-hour light-and-dark cycle. Food and water wereavailable ad libitum.

MDA-MB-231 human mammary carcinoma cells were grown and maintained inculture using Leibovits media with 10% fetal bovine serum. The cellswere kept at 37° C. in a humidified incubator. Log-phase cells weretrypsinized and harvested from culture flasks and centrifuged at 900 rpmfor 10 minutes. The supernatant was discarded and cell pelletre-suspended in Hank's Balanced Salt Solution (HBSS) at 10×10⁷ cells/mL(NB 7301 page 112). The cell suspension was then injected subcutaneouslyin 100 μL to yield an inoculum of 10×10⁶ cells. The mean tumor size attime of treatment initiation were approximately 150 mm³.

3. Study Design

Treatment groups are summarized in Table 9. Ten animals were assigned toeach treatment group. All formulations were administered intravenously(IV) into the lateral tail veins of mice restrained in a heated (40° C.)brass. Immediately prior to each injection, mice were kept warm in awell-ventilated acrylic box with a heating light bulb (ALZA SOP 8-650).Doxorubicin dose was either 1 or 4 mg/kg given once weekly for 4 weeks.

TABLE 9 Study Design for Evaluation of Antitumor Activity of □_(v)Targeted STEALTH ® Liposomal Doxorubicin in MDA-MB-231 Human MammaryCarcinoma Xenografts Fab′/Liposome Dose Route No. of Treatment Groupsratio (mg/kg) of Inj. Dosing Days Mice 1. Untreated Control N/A N/A — —10 2. S-DOX 0 1 IV Day 0, 7, 14, 21 10 3. Fab′-S-DOX 15:1 1 IV Day 0, 7,14, 21 10 4. Fab′-S-DOX 40:1 1 IV Day 0, 7, 14, 21 10 5. Fab′-S-DOX 90:11 IV Day 0, 7, 14, 21 10 6. S-DOX 0 4 IV Day 0, 7, 14, 21 10 7.Fab′-S-DOX 15:1 4 IV Day 0, 7, 14, 21 10 8. Fab′-S-DOX 40:1 4 IV Day 0,7, 14, 21 10 9. Fab′-S-DOX 90:1 4 IV Day 0, 7, 14, 21 10

Tumors were measured in three dimensions up to 3 times weekly until theaverage tumor volume for a treatment group reached 1000 mm³ or up to 60days. Tumor volume was calculated according to the formula:

V=½×D ₁ ×D ₂ ×D ₃

where D₁₋₃ are perpendicular diameters measured in millimeters (mm).

Animal body weights were measured three times a week to assess drugtoxicity. Clinical observations included behavior or activity within thecage, and signs of pain or distress. All abnormal clinical observationswere recorded either in the comment section of the data sheets or theevent log. At the end of study period, all animals were providedeuthanasia by inhalation of 100% carbon dioxide according to the AVMAPanel on Euthanasia (1993).

Statistical analysis of tumor growth between the various treatmentgroups was performed by one-way analysis of variance supplemented with aTukey's multiple comparison post-test (Days 7 to 60) and by Kaplan-MeierLog Rank test (endpoint: time to reach 1000 mm³). Both test were doneusing GraphPad Prism® 4 software.

4. Results

This study evaluated the antitumor activity of α_(v)-targeted liposomaldoxorubicin administered IV once weekly for four weeks on MDA-MB-231human mammary carcinoma xenografts. Tumor growth curves and changes inbody weights as a function of time are presented in FIGS. 12A-12B. Thetumor growth and body weight are reported as a percent change relativeto the initial tumor size and weight for each animal. FIG. 12C shows thesurvival data for the animals in each treatment group.

The average time for tumor growth to 1000 mm³ volume was 15.2±0.9 daysfor untreated controls. Tumor in animals treated with S-DOX at 1 and 4mg/kg reached 1000 mm³ in 25.8±5.5 and 54.8±2.5 days, respectively. Daysto 1000 mm³ endpoint for Fab′-S-DOX (15:1, 40:1 and 90:1) treatmentgroup were 29.7±5.2, 34.9±4.3, and 24.6±4.2 days at 1 mg/kg, and59.1±0.7, 50.8±3.3, and 53.4±3.8 days at 4 mg/kg, respectively. Fortumors that had not reached the study endpoint by Day 60, 60-days wasused in the data analysis.

No animal body weight loss was observed in all treatment groups. Oneanimal in Fab′-S-DOX (90:1) treatment group at 4 mg/kg was found dead onDay 25.

One way analysis of variance (Days 7 to 60) supplemented with Tukey'spost test indicated that all groups treated with 1 mg/kg doxorubicinwere similar to untreated control, and those treated with 4 mg/kgformulations had significantly greater antitumor activity than untreatedcontrol. The Kaplan-Meier Log Rank Test (endpoint: time to 1000 mm³)indicated a significant antitumor activity for all doxorubicin treatmentgroups except for S-DOX at 1 mg/kg. However, at each drug dose level,there was no difference between the targeted and non-targetedformulation or between targeted formulations containing different liganddensity.

In this study using the MDA-MB-231 human mammary carcinoma xenograftmodel, similar antitumor activity was observed for S-DOX and Fab′-S-DOX(15:1, 40:1 and 90:1) formulations at the same dose level (1 or 4mg/kg).

Example 8 Antitumor Activity of AV-Integrin-Targeted Liposomes

This study investigated the ability of integrin-targeted immunoliposomescontaining entrapped doxorubicin to inhibit growth of A375S.2 humanmelanoma tumors in rats.

1. Liposome Compositions

Liposomes lacking an integrin targeting ligand, referred to as “SLD”,were prepared as described in Example 1.

Integrin-targeted liposomes, referred to as “ITL”, were also prepared asdescribed in Example 1 and 2. Integrin-targeted immunoliposomeformulations having 15:1 and 30:1 Fab′ to liposome ratios were prepared.The 15:1 formulation had a doxorubicin concentration of 2.23 mg/mL, andthe 30:1 formulation had a doxorubicin concentration of 2.26 mg/mL.

2. Xenograft Preparation

Female nude rats approximately 6-8 weeks of age were obtained (HarlanLaboratories, Indianapolis, Ind.). The rats were group-housed (2/cage)in filter-topped plastic cages and supplied with autoclaved food andwater. Each animal was tail tattooed with a number or ear tagged priorto the start of the study.

A375S.2 human melanoma tumor cells, free of bacteria and mycoplasma,were cultured in DMEM containing Glutamax, 10% FBS, and 1% non-essentialamino acids (complete medium). On the day of the study initiation, cellswere trypsinized to generate a single cell suspension, then spun downand resuspended in serum-free DMEM. The final concentration of the cellsuspension was 2.5×10⁷ cells/mL.

On day 0, 90 female nude rats were treated with one chewable antibiotictablet containing 10 mg Trimethoprim and 60 mg sulfamethoxazole (SCID'sMD sterile bacon-flavored, Bio-Serv) per cage, once daily. Antibiotictreatment began three to four days before irradiation (Day −5) andcontinued for two weeks. One day before tumor implantation (Day −1), theanimals received 2 rads of whole body irradiation per 1 gram bodyweight. On Day 0, rats were implanted with 0.2 mL of A375S.2 cellsuspension as described above.

Rats were monitored twice a week for appearance of a palpable tumor.When 70 rats had tumors that measured approximately 50-250 mm³ (Day 8),they were stratified into seven groups with 10 animals each, fortreatment as set forth in Table 10. Day 8 was the start of treatment.Rats were weighted on the day of drug dosing, and were injected iv atweekly intervals for four total doses with 2 or 0.5 mg/kg of testliposome formulation or were given a saline control.

Tumor growth was measured twice a week with calipers in two dimensions(length and width) in millimeters (mm). Tumor volume (mm³) wascalculated based on the formula (length×width×width)/2.

TABLE 10 Treatment Groups Doxorubicin Dose Concentration Group Treatment(mg/kg) (mg/mL) 1 PBS 1 mL/100 g 0 body weight 2 SLD 2 0.2 3 ITL 15:1 20.2 4 ITL 30:1 2 0.2 5 SLD 0.5 0.05 6 ITL 15:1 0.5 0.05 7 ITL 30:1 0.50.05

Tumor weight data was analyzed via standard linear model and analysis ofvariance (ANOVA). P-values less than 0.05 for all tests and comparisonswere deemed significant unless otherwise indicated. A logarithmic scalewas used since underlying assumptions of equal variance and normaldistribution shape were better satisfied. The zero and 0.5 values, formice that measured little to no tumor, were replaced with smallspline-interpolated value that facilitated statistical analysis in thelogarithmic scale without corruption of the data structure.

For the tumor volume, a repeated measures model was fit to the dataassuming a first order autocorrelation covariance structure. Naturalsplines were used to model the curvature of trends in the time profiles.Pairwise correlations amongst the groups were made at each of thetimepoints. Calculations were performed using the R softwareenvironment.

3. Results

The immunoliposome formulations having 15:1 and 30:1 anti-integrin Fab′antibodies per liposomes were administered once a week for four doses,beginning when the tumors were approximately 165 mm³. Tumor growthcurves are shown in FIGS. 13A-13B, for rats treated with 2 mg/kg and 0.5mg/kg doxorubicin, respectively. The negative control group, PBS treatedanimals, shows complete tumor take and steady tumor growth, with thegroup reaching maximal tumor volume in 29 dyas. All rats treated withdoxorubicin entrapped in liposomes lacking a targeting ligand (“SLD”) orbearing a targeting ligand (“ITL”) showed significantly delayed tumorgrowth. The 0.5 mg/kg groups reached maximal tumor volume on Day 39 andthe 2 mg/kg groups reached maximal tumor volume on Day 49.

The immunoliposomes bearing 15:1 Fab′ fragments per liposome showed atrend of tumor growth delay when compared to liposomes lacking atargeting ligand. Accordingly, in a preferred embodiment,immunoliposomes bearing fewer than about 25 targeting ligand perliposome, preferably fewer than 20, and still more preferably about 15or fewer targeting ligands per liposome, is contemplated.

Example 9 Generation of a Host Cell Line Producing Anti-Alpha-V Fab

The CNTO95 heavy chain signal peptide and variable region from SEQ IDNO: 1 were cloned into expression vector p2032. This vector contained amouse immunoglobulin promoter, a human IgG1 CH₁ constant region, thefirst cysteine in the a human IgG1 hinge sequence followed by PGK, and aGPT gene for selection of stable integration into the host cell genome.The completed CNTO95 heavy chain Fab expression plasmid, p2324, encodingSEQ ID NO: 3, was co-transfected with the CNTO95 light chain expressionplasmid, p2330, into sp2/0 mouse myeloma cells. Cell clones with stablegenomic integration of the plasmids were selected based on theirresistance to mycophenolic acid in the presence of hypoxanthine. Theseclones were assayed for Fab expression by ELISA and western blot. Thehighest expressing clones were subjected to one round of subcloning,with the best subclone expressing 10 ug/ml. This clone, C1021A, wasscaled up for further analysis.

The resulting Fab product was designated CNTO 119 and comprised SEQ IDNO: 3 and SEQ ID NO: 2). The C-terminus of the heavy chain bears asingle cysteine which can be used effectively for conjugation reactionsfollowing mild reduction. The three C-terminal amino acids (PGK) are thesame as the C-terminal residues of the full-length IgG1 heavy chain(including CNTO95 heavy chain).

Example 10 Preparation of Conjugated sFab-Targeted STEALTH Liposomes

A significant portion of the sFab starting material was in the oxidizeddisulfide form and was subjected to reduction using 10 mM DTE, 40° C.,pH 6.0 for 60 minutes to form a free sulfhydryl for conjugation. Excessreductant was removed by passing the reduced sFab material over adesalting column using saline as the running buffer. The pH of thecollected sFab fraction was adjusted to pH 6.0 and the proteinconcentration measured. Since the reduction process produces significantamounts of unwanted by-products (i.e., unassociated light and heavychains), the sFab material was then subjected to oxidation byintroduction of oxygen into the solution to reform the criticaldisulfide bond between the light and heavy chains that form sFab.Reformation of sFab was monitored by SEC-HPLC. After approximately 4.5hours of the oxidation reaction, the sFab material was conjugated withMalPEG-DSPE (5:1 MalPEG-DSPE:sFab ratio) at room temperature for 1 hour.The solution was quenched for 10 minutes using 1 mM cysteine and runover a desalting column to remove unreacted cysteine. The resultingconjugated sFab material was loaded onto a SEC column (Sephacryl 300) toremove unconjugated protein with PBS as the running buffer. Theresulting purity and yield of sFab-conjugate was 95% and 67%,respectively.

sFab-conjugate material was placed over a desalting column to exchangethe external buffer to saline. Liposomes containing encapsulateddoxorubicin, as described in Example 1 in saline, were inserted withsFab-conjugate at either 60° C. for 1 hour or 37° C. for 48 hour. Theamount of sFab added to the liposomes was sufficient to achieve thedesired ratio of 15 sFab ligands per liposome. After insertion, the sFabliposome solution was diluted to a final target concentration of 2 mg/mLwith saline. The final formulations were in saline. sFab liposomesamples were only tested on confocal microscopy to assay bioactivity.

Bioactivity results were negative for formulations inserted at 60° C.while marginal bioactivity of the formulations inserted at 37° C. wasobserved.

Example 11 Preparation of a Single Chain Anti-Alpha-V Antibody

1. scFv Engineering

Single chain variable fragments (scFv) of antibodies are well-suited astargeting agents due to their small size and compatibility with thecysteine-based PEG coupling chemistry required to incorporate thetargeting agent into the STEALTH liposome. To this end, scFv derivativesof CNTO 95 were designed, engineered and expressed. Several variantswere designed in order to overcome problems with expression. In onevariant, the naturally occurring Arg-Arg amino acid pair, whichfrequently inhibits expression in E. coli, was mutated to Leu-Arg. Theleucine substitution (R18L, an arg mutation to leu at the 18^(th) aminoacid of HC variable region) resulted in a substantial improvement ofexpression in E. coli cells using an arabinose inducible promoter (Xomasystem, Xoma LLC, Berkeley, Calif.).

CNTO95 scFv variable heavy and light chain sequences were derived fromCNTO95 Mab (U.S. Pat. No. 7,163,681). A flexible linker (Gly4Ser)₃connects the variable regions to provide sufficient conformationalflexibility for the pairing of the VH and VL domains of SEQ ID NO: 1residues 1-119 and 2 residues 1-108, respectively. The construct wasexpressed transiently in HEK293 cells. The E. coli constructs wereoptimized for expression by engineering the codons that are consideredrare in E. coli to more frequently used synonymous codons. Allconstructs contain a PelB signal sequence and a C-terminal 6×His tagfollowed by a Gly4Cys to allow purification and PEG conjugation toliposomes, respectively (SEQ ID NO: 5). The R18L derivative proteincoding region was designed to be compatible with expression vectorsestablished for other scFv such as the F5, directed against human ErbB2selected from a scFv phage library (WO99/55367 and WO99/56129) and whichall comprised the (G4S)₃ linker. The resulting EcoRI and XhoI fragmentwas cloned into the pING3302 vector (Xoma), which vector has anarabinose inducible gene for expression and tetracycline resistant geneand is related to a previously described vector for Fab expression(Chowdhury, P. S., I. Pastan. 1999. Nat. Biotechnol. 17:568-572).Multiple variants of this construct series were generated to optimizethe expression level of the scFv constructs, these included theconstruct without a His tag, or with a (G₄S)₄ linker, or with alternateheavy and light chain framework regions as shown in Table 11.

TABLE 11 Plasmid Expression Constructs 4016 scFv CNTO 95 (EG0001).PelB-Vh-(G4S)x3-Vk-G4C, 4017 scFv CNTO 95 (EG0002).PelB-Vh-(G4S)x4-Vk-G4C 4018 scFv CNTO 95 (EG0003).PelB-Vh-(G4S)x3-Vk-His6tag-G4C 4028 scFv CNTO 95 (CNTBH117)PelB-Vh-(G4S)x3-Vk 3202 scFv F5 with histag (CNTOBH 120) in XOMA vector.3203 scFv F5 with histag and Cys (CNTOBH 121) in XOMA vector.. 3204 scFvCNTO95 with (G4S)X3, histag and Cys (CNTOBH 122) in XOMA vector. 4071scFv CNTO95 R18L (P3204). Change RR to LR on HC V-region 4073 scFvCNTO95 with (G4S)X4 linker but is otherwise identical to P3204 4072 scFvCNTO95 R18L (P4073). Change RR to LR on HC V-region 4225 scFv CNTO95R18L (P4071) without Cys

2. ScFv CNTO95 Expression

For mammalian expression, HEK 293 cells were transiently transfected inserum-free media using the mammalian expression vector pCEP4(Invitrogen), previously modified to contain the CMV-IE intron A. After4 days, conditioned supernatant was tested for scFv protein expressionby Western blot and ELISA. The plasmid comprising the CNTO95 scFv HCOR18L in pBeth vector was designated p4544.

For E. Coli, expression was performed using an arabinose induciblesystem obtained from Xoma, Inc. The DNA was transformed into E. coliE104 competent cells (competent cells were prepared by following astandard CaCl₂ solution method (T. Maniatis et al., 1982, MolecularCloning (A Laboratory Manual), Cold Spring Harbor Laboratory). A singlecolony was grown in 2 ml 2×YT medium with 25 ug/ml tetracycline at 37°C. overnight. The culture (diluted 1/500) was expanded in 250 ml 2×YTmedium with 25 ug/ml tetracycline. After the culture reached OD600 at0.6, it was induced with 0.1% of L-arabinose at 25° C. overnight andharvested by centrifugation at 10 K for 15 min. The supernatant was usedto detect the expression of the expected protein by Western blotting.The pellet was lysed with 20 ml B-PERII bacterial protein extractionbuffer (Pierce), followed by centrifugation, and the resultingsupernatant was processed to purify the induced protein using Talonresin (BD Biosciences), according the manufacturer's instruction.

Initial expression in the Xoma system produced very low levels of scFvprotein. Tandem arginine residues were identified in the FR1 region ofthe heavy chain at positions 18 and 19. Since double arginine amino acidresidues have been shown to be potentially detrimental for proteinexpression, arginine 18, the first arginine in the doublet, was revertedto leucine, LR, which corresponds to the human VH germline sequencehaving the closest homology and derived from Ig H-chain V-region (DP-46)(Referenced as NCBI Accession No. CAA78216) and as shown below. The 28kD scFv protein was only detected in E. coli supernatant fractions whenvariants with the R18L mutation were expressed.

3. ScFv CNTO95 Detection

Supernatant, cell lysate and purified protein were separated byelectrophoresis on 4-12 SDS-polyacrylamide gels and transferred to PVDFmembranes. The membranes were blocked with 5% nonfat dry milk in TBScontaining 0.05% Tween-20 (wash buffer) at room temperature for 1 hr.

To detect scFv protein, two distinct antibodies were used for Westernblotting. The first one was anti-his antibody, while the second one wasanti-CNTO95 idiotypic antibody (C508, murine anti-CNTO95), which hasbinding affinity to CNTO95 variable domains. For anti-his Western blot,the membrane was incubated with peroxidase-conjugated anti-His antibody(1:5000 dilution), and the his-tag protein was detected using ECLWestern Blotting Analysis System (Amersham Biosciences). For the antiCNTO95 id Western blot, the capture antibody was C508 antibody (1:1000dilution). Peroxidase-conjugated anti-mouse antibody (1:5000 dilution)was used as the secondary antibody, followed by detection of boundprotein using the ECL Western Blotting Analysis System (AmershamBiosciences). Using the anti-his antibody as a probe, the Western Blotshowed that the 28 kD scFv protein was only detected in variants withthe R18L mutation.

4. scFv Binding Affinity:

The binding affinity of scFv CNTO95 was measured using a validatedsandwich enzyme-linked immunoassay. The 96-well plates (Coasta, highbinding plate) were coated with 100 ul of integrin αvβ3 or αvβ5(Chemicon International Inc ) at 1 ug/ml in coating buffer (0.75 gNa₂CO₃ and 1.45 g NaHCO₃ in 500 ml H₂O) and incubated overnight at 4° C.The plates were blocked with Superblock blocking buffer in PBS (Pierce)at room temperature for 1 hr. Wells were washed with PBS+0.05% Tween 20between each step. Controls and scFv purified protein were dilutedserially at 3-fold with TBST, and added to the coated wells induplicate, and incubated at 37° C. for 2 hrs. Two methods were used todetect the binding. For anti-his ELISA 1:1000 diluted HRP mouse anti-hisantibody were added to each well and incubated for 1 hr. For theanti-CNTO95 anti-idiotype ELISA, an aliquot of 1:1000 diluted 1 mg/mlC508 antibody was added to each well and incubated for 1 hr.Peroxidase-conjugated anti-mouse antibody (1:5000 dilution) was used asthe secondary antibody, followed by incubation at the plate for 1 hr at37° C.

The Elisa was developed using OPD tablet (Sigma) in development bufferfor 15 min at room temperature. The colorimetric detection was stoppedwith 1N H₂SO₄. The binding affinity was measured in a plate reader at490 nm. In the assay, purified CNTO95 scFv protein from E. coli boundboth αvβ3 and αvβ5 integrin proteins (FIG. 14) as shown forintegrin-coated ELISA plates and was detected by HRP anti-His Ab as wellas the anti-CNTO95 Ab (c508).

ScFv vs Mab Binding Affinity to Integrin Competition Assay:

To compare the binding affinity of scFv CNTO95 and CNTO 95 mAb, the96-well plates were coated with 100 ul of integrin αvβ3 or αvβ5 asdescribed above. CNTO95 mAb was added to each well at 0.8 nM for αvβ3coated plate or 0.2 nM for αvβ5 coated plate. Controls and scFv purifiedprotein diluted serially at 3-fold with TBST were added to the coatedwells in duplicate. The mix of CNTO95 mAb and sFv were incubated at 37°C. for 2 hrs. The detection antibody was 1:10K dilution of peroxidaseconjugated affinipure F(ab′)₂ fragment Goat anti-human IgG, Fc fragmentspecific (minimal cross reaction to Bovine, Horse and mouse serumprotein) (Jackson Immunoresearch ). The antibody was incubated on theplate 1 hr at 37° C. and the color was developed and read as above.CNTO95 mAb binding to both alpha-V-integrins was competed with the CNTO99 scFv (FIGS. 15A and B). The assay indicates CNTO95 scFv effectivelycompetes with the Mab for integrins binding in a manner comparable tothe F(ab)′2 protein.

Summary

The optimized CNTO95 scFv is composed of an immunoglobulin heavy-chainleader sequence and heavy and light chain variable regions that arejoined by an inter-chain (Gly₄Ser)₃ linker which allows conformationalflexibility. A Pel B signal sequence was placed upstream of the antibodycoding sequence with in the vector to facilitate secretion of theantibody. The E. coli constructs were further optimized for expressionby engineering the codons that are considered rare in E. coli to morefrequently used synonymous codons. All constructs contain a C-terminal6×His tag, to facilitate purification, followed by a Gly₄Cys to allowchemical conjugation via the free sulfhydryl to, e.g. a thiol-reactivederivatized PEG, for insertion into liposomes. The disruption of anarginine doublet in the heavy chain sequence (18-19) by the R18Lmutation proved essential for secretion and recover of the 28 kD scFvprotein. Variants with different linker lengths did not alter expressionlevels.

Example 12 Preparation of Conjugated scFv-Targeted STEALTH Liposomes

As a significant portion of the scFv starting material was oxidized andin the dimeric (disulfide form) the scFv was subjected to reductionusing 10 mM DTE, 30° C., pH 7.0 for 60 minutes to form a free sulfhydrylfor conjugation. Excess reductant was removed by passing the scFv over adesalting column using saline as the running buffer. The pH of thecollected scFv fraction was adjusted to pH 6.0 and the proteinconcentration measured.

Liposomes containing encapsulated doxorubicin, as described in Example1, were inserted with maleimide-PEG-lipid (MalPEG-DSPE) at approximately800 MalPeg-DSPE per liposome at 60° C. for 1 Hr. The appropriate amountof scFv was added to the MalPEG-DSPE inserted liposomes to achieve thedesired ratio (15, 40 or 90 to 1). It was assumed that 50% of the scFvwould conjugate to the liposomes based on previous work. The conjugationproceeded at room temperature for 2 hours followed by overnightrefrigeration. Each formulation was quenched by adding 1 mM Cysteine for10 min and passed over a size-exclusion column to remove nonconjugatedscFv (monomer and dimer) and free cysteine. The final formulations werein saline, 10 mM histidine at pH 6.5.

Example 13 Cytotoxicity Assays Using scFv-Targeted STEALTH Liposomes

Human melanoma cells (A375.S2, ATCC CRL 1872), passage 3 to 9, wereincubated in EMEM (Eagle's Minimum Essential Medium, ATCC Cat No.30-2003) containing 10% FCS (ATCC Cat No. 30-2021). On the day of theassay, cells were scraped from the surface of the culture flask andpipetted up and down using serum containing media to make single cellsuspension. The cells were rejuvenated by suspending them in completemedium at 37° C. for 1 hour with mild shaking. Thereafter, the cellswere pelleted and resuspended in serum-free medium at 200 K in 1 ml in a5 ml polypropylene round-bottom tube (BD Falcon).

Freshly prepare anti-alpha-V scFv (CNTO99) targeted DOXIL liposomesprepared with varying ratios of antibody to liposome: 15:1, 40:1, 90:1were diluted by serial 5-fold dilutions (1.6, 8, 40 and 200 ug/ml ofDOXIL as doxorubicin with a prewarmed (37° C.) solution of 10%sucrose+ions (1 mM CaCl2, 1 mM MgCl2, 10 uM MnCl2), pH 5.7. UntargetedDOXIL and doxorubicin (DXR) was prepared similarly. Untreated group:A375.S2 cell only be incubated with diluting solution, 10% sucrose+ionspH 5.7, at 37° C.

To the respective labeled tubes containing the cell suspensions, wasadded 0.1 ml of scFv-targeted DOXIL, DOXIL, or DXR treatment solution.The tubes were shaken to mix and incubated at 37° C. for 10 min, withmild agitation (140 rpm on an orbital shaker). Treatments were stoppedby adding 1.0 ml of 37° C. cell culture medium without FBS, vortexingbriefly, and pelleting the cells at room temperature. The supernatantwas discarded and cells resuspended in 1 ml of 37° C. cell culturemedium without FBS. Washing was repeated twice and the cells finallyresuspended in 2.0 mL of cell culture medium with FBS (37° C.). Thecells were then counted and seeded at 2,000 cells/200 μl into each wellof the 96 well plate. The plates were incubated at 37° C. for 3 days and6 days at which time the cell number was quantitated using a CellTiter96®AQueous One Solution (Promega) according to the manufacturersinstructions using a Spectra Max 250, Molecular Devices spectrometer setat 490 nm.

The results shown in FIG. 16 indicate that liposomes with greater than15 scFv as targeting ligand on the surface are more effective in killingtumor cells (or preventing tumor cell growth) than untarget liposomes orfree doxorubicin.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

1. An anti-alpha-V targeted immunoliposome composition, comprisingliposomes comprised of vesicle-forming lipids and a conjugate comprisedof a hydrophobic moiety, a hydrophilic polymer, and an anti-alpha-Vantibody-derived construct having binding affinity for alpha V integrinsubunit, said liposomes prior to in vivo administration having fewerthan 25 antibodies per liposome.
 2. The composition of claim 1, whereinsaid liposome having an entrapped drug.
 3. The composition of claim 2,wherein said drug is an anthracycline antibiotic.
 4. The composition ofclaim 3, wherein said anthracycline antibiotic is doxorubicin.
 5. Thecomposition of claim 1, wherein said liposomes prior to in vivoadministration having about 15 antibodies per liposome.
 6. A method oftreating a condition characterized by cells that express at least onealpha-V-integrin receptor, comprising: administering immunoliposomesaccording to claim
 1. 7. The method of claim 6, wherein said conditionis characterized by cells that express one or both of αvβ3 and α5β5 andwhich condition is a neoplastic disease.
 8. The method of claim 7,wherein said neoplastic disease is melanoma.
 9. The method of claim 6,wherein said administering comprises administering immunoliposomesfurther comprising an entrapped therapeutic agent.